Methods and compositions for detecting target sequences

ABSTRACT

The present invention relates to compositions and methods for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. The present invention relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. For example, in some embodiments, a 5′ nuclease activity from any of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

[0001] The present Application claims priority to U.S. ProvisionalApplication 60/344,946 filed Nov. 7, 2001, and to U.S. ProvisionalApplication 60/361,060 filed Feb. 27, 2002, each hereby incorporated byreference in their entireties.

[0002] This Application is a continuation-in-part of co-pendingapplication Ser. No. 09/713,601, which is a continuation-in-part of U.S.Pat. No. 6,348,314, which is a divisional of U.S. Pat. No. 5,985,557,and is also a continuation-in-part of co-pending application Ser. No.09/381,212, which is a national entry of PCT Application No. U.S.98/05809, which claims priority to U.S. Pat. Nos. 5,994,069, 6,090,543,5,985,557, 6,001,567, and 5,846,717 and PCT Application No. U.S.97/01072.

FIELD OF THE INVENTION

[0003] The present invention relates to compositions and methods for thedetection and characterization of nucleic acid sequences and variationsin nucleic acid sequences. The present invention relates to methods forforming a nucleic acid cleavage structure on a target sequence andcleaving the nucleic acid cleavage structure in a site-specific manner.For example, in some embodiments, a 5′ nuclease activity from any of avariety of enzymes is used to cleave the target-dependent cleavagestructure, thereby indicating the presence of specific nucleic acidsequences or specific variations thereof.

BACKGROUND OF THE INVENTION

[0004] Methods for the detection and characterization of specificnucleic acid sequences and sequence variations have been used to detectthe presence of viral or bacterial nucleic acid sequences indicative ofan infection, to detect the presence of variants or alleles of genesassociated with disease and cancers. These methods also find applicationin the identification of sources of nucleic acids, as for forensicanalysis or for paternity determinations.

[0005] Various methods are known to the art that may be used to detectand characterize specific nucleic acid sequences and sequence variants.Nonetheless, with the completion of the nucleic acid sequencing of thehuman genome, as well as the genomes of numerous pathogenic organisms,the demand for fast, reliable, cost-effective and user-friendly testsfor the detection of specific nucleic acid sequences continues to grow.Importantly, these tests must be able to create a detectable signal fromsamples that contain very few copies of the sequence of interest. Thefollowing discussion examines two levels of nucleic acid detectionassays currently in use: I. Signal Amplification Technology fordetection of rare sequences; and II. Direct Detection Technology forquantitative detection of sequences.

[0006] I. Signal Amplification Technology Methods for Amplification

[0007] The “Polymerase Chain Reaction” (PCR) comprises the firstgeneration of methods for nucleic acid amplification. However, severalother methods have been developed that employ the same basis ofspecificity, but create signal by different amplification mechanisms.These methods include the “Ligase Chain Reaction” (LCR), “Self-SustainedSynthetic Reaction” (3SR/NASBA), and “Qβ-Replicase” (Qβ).

[0008] Polymerase Chain Reaction (PCR)

[0009] The polymerase chain reaction (PCR), as described in U.S. Pat.Nos. 4,683,195, 4,683,202, and 4,965,188 to Mullis and Mullis et al.(the disclosures of which are hereby incorporated by reference),describe a method for increasing the concentration of a segment oftarget sequence in a mixture of genomic DNA without cloning orpurification. This technology provides one approach to the problems oflow target sequence concentration. PCR can be used to directly increasethe concentration of the target to an easily detectable level. Thisprocess for amplifying the target sequence involves introducing a molarexcess of two oligonucleotide primers that are complementary to theirrespective strands of the double-stranded target sequence to the DNAmixture containing the desired target sequence. The mixture is denaturedand then allowed to hybridize. Following hybridization, the primers areextended with polymerase so as to form complementary strands. The stepsof denaturation, hybridization, and polymerase extension can be repeatedas often as needed, in order to obtain relatively high concentrations ofa segment of the desired target sequence.

[0010] The length of the segment of the desired target sequence isdetermined by the relative positions of the primers with respect to eachother, and, therefore, this length is a controllable parameter. Becausethe desired segments of the target sequence become the dominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR-amplified.”

[0011] Ligase Chain Reaction (LCR or LAR)

[0012] The ligase chain reaction (LCR; sometimes referred to as “LigaseAmplification Reaction” (LAR) described by Barany, Proc. Natl. Acad.Sci., 88:189 (1991); Barany, PCR Methods and Applic., 1:5 (1991); and Wuand Wallace, Genomics 4:560 (1989) has developed into a well-recognizedalternative method for amplifying nucleic acids. In LCR, fouroligonucleotides, two adjacent oligonucleotides which uniquely hybridizeto one strand of target DNA, and a complementary set of adjacentoligonucleotides, that hybridize to the opposite strand are mixed andDNA ligase is added to the mixture. Provided that there is completecomplementarity at the junction, ligase will covalently link each set ofhybridized molecules. Importantly, in LCR, two probes are ligatedtogether only when they base-pair with sequences in the target sample,without gaps or mismatches. Repeated cycles of denaturation,hybridization and ligation amplify a short segment of DNA. LCR has alsobeen used in combination with PCR to achieve enhanced detection ofsingle-base changes. Segev, PCT Public. No. W09001069 A1 (1990).However, because the four oligonucleotides used in this assay can pairto form two short ligatable fragments, there is the potential for thegeneration of target-independent background signal. The use of LCR formutant screening is limited to the examination of specific nucleic acidpositions.

[0013] Self-Sustained Synthetic Reaction (3SR/NASBA)

[0014] The self-sustained sequence replication reaction (3SR) (Guatelliet al., Proc. Natl. Acad. Sci., 87:1874-1878 [1990], with an erratum atProc. Natl. Acad. Sci., 87:7797 [1990]) is a transcription-based invitro amplification system (Kwok et al., Proc. Natl. Acad. Sci.,86:1173-1177 [1989]) that can exponentially amplify RNA sequences at auniform temperature. The amplified RNA can then be utilized for mutationdetection (Fahy et al., PCR Meth. Appl., 1:25-33 [1991]). In thismethod, an oligonucleotide primer is used to add a phage RNA polymerasepromoter to the 5′ end of the sequence of interest. In a cocktail ofenzymes and substrates that includes a second primer, reversetranscriptase, RNase H, RNA polymerase and ribo-and deoxyribonucleosidetriphosphates, the target sequence undergoes repeated rounds oftranscription, cDNA synthesis and second-strand synthesis to amplify thearea of interest. The use of 3SR to detect mutations is kineticallylimited to screening small segments of DNA (e.g., 200-300 base pairs).

[0015] Q-Beta (Qβ) Replicase

[0016] In this method, a probe that recognizes the sequence of interestis attached to the replicatable RNA template for Qβ replicase. Apreviously identified major problem with false positives resulting fromthe replication of unhybridized probes has been addressed through use ofa sequence-specific ligation step. However, available thermostable DNAligases are not effective on this RNA substrate, so the ligation must beperformed by T4 DNA ligase at low temperatures (37° C.). This preventsthe use of high temperature as a means of achieving specificity as inthe LCR, the ligation event can be used to detect a mutation at thejunction site, but not elsewhere.

[0017] Table 1 below, lists some of the features desirable for systemsuseful in sensitive nucleic acid diagnostics, and summarizes theabilities of each of the major amplification methods (See also,Landgren, Trends in Genetics 9:199 [1993]).

[0018] A successful diagnostic method must be very specific. Astraight-forward method of controlling the specificity of nucleic acidhybridization is by controlling the temperature of the reaction. Whilethe 3SR/NASBA, and Qβ systems are all able to generate a large quantityof signal, one or more of the enzymes involved in each cannot be used athigh temperature (i.e., >55° C.). Therefore the reaction temperaturescannot be raised to prevent non-specific hybridization of the probes. Ifprobes are shortened in order to make them melt more easily at lowtemperatures, the likelihood of having more than one perfect match in acomplex genome increases. For these reasons, PCR and LCR currentlydominate the research field in detection technologies. TABLE 1 MethodPCR & 3SR Feature PCR LCR LCR NASBA Qβ Amplifies Target + + + +Recognition of Independent + + + + + Sequences Required Performed atHigh Temp. + + Operates at Fixed Temp. + + ExponentialAmplification + + + + + Generic Signal Generation + Easily Automatable

[0019] The basis of the amplification procedure in the PCR and LCR isthe fact that the products of one cycle become usable templates in allsubsequent cycles, consequently doubling the population with each cycle.The final yield of any such doubling system can be expressed as:(1+X)^(n)=y, where “X” is the mean efficiency (percent copied in eachcycle), “n” is the number of cycles, and “y” is the overall efficiency,or yield of the reaction (Mullis, PCR Methods Applic., 1:1 [1991]). Ifevery copy of a target DNA is utilized as a template in every cycle of apolymerase chain reaction, then the mean efficiency is 100%. If 20cycles of PCR are performed, then the yield will be 2²⁰, or 1,048,576copies of the starting material. If the reaction conditions reduce themean efficiency to 85%, then the yield in those 20 cycles will be only1.85²⁰, or 220,513 copies of the starting material. In other words, aPCR running at 85% efficiency will yield only 21% as much final product,compared to a reaction running at 100% efficiency. A reaction that isreduced to 50% mean efficiency will yield less than 1% of the possibleproduct.

[0020] In practice, routine polymerase chain reactions rarely achievethe theoretical maximum yield, and PCRs are usually run for more than 20cycles to compensate for the lower yield. At 50% mean efficiency, itwould take 34 cycles to achieve the million-fold amplificationtheoretically possible in 20, and at lower efficiencies, the number ofcycles required becomes prohibitive. In addition, any backgroundproducts that amplify with a better mean efficiency than the intendedtarget will become the dominant products.

[0021] Also, many variables can influence the mean efficiency of PCR,including target DNA length and secondary structure, primer length anddesign, primer and dNTP concentrations, and buffer composition, to namebut a few. Contamination of the reaction with exogenous DNA (e.g., DNAspilled onto lab surfaces) or cross-contamination is also a majorconsideration. Reaction conditions must be carefully optimized for eachdifferent primer pair and target sequence, and the process can takedays, even for an experienced investigator. The laboriousness of thisprocess, including numerous technical considerations and other factors,presents a significant drawback to using PCR in the clinical setting.Indeed, PCR has yet to penetrate the clinical market in a significantway. The same concerns arise with LCR, as LCR must also be optimized touse different oligonucleotide sequences for each target sequence. Inaddition, both methods require expensive equipment, capable of precisetemperature cycling.

[0022] Many applications of nucleic acid detection technologies, such asin studies of allelic variation, involve not only detection of aspecific sequence in a complex background, but also the discriminationbetween sequences with few, or single, nucleotide differences. Onemethod for the detection of allele-specific variants by PCR is basedupon the fact that it is difficult for Taq polymerase to synthesize aDNA strand when there is a mismatch between the template strand and the3′ end of the primer. An allele-specific variant may be detected by theuse of a primer that is perfectly matched with only one of the possiblealleles; the mismatch to the other allele acts to prevent the extensionof the primer, thereby preventing the amplification of that sequence.This method has a substantial limitation in that the base composition ofthe mismatch influences the ability to prevent extension across themismatch, and certain mismatches do not prevent extension or have only aminimal effect (Kwok et al., Nucl. Acids Res., 18:999 [1990]).)

[0023] A similar 3′-mismatch strategy is used with greater effect toprevent ligation in the LCR (Barany, PCR Meth. Applic., 1:5 [1991]). Anymismatch effectively blocks the action of the thermostable ligase, butLCR still has the drawback of target-independent background ligationproducts initiating the amplification. Moreover, the combination of PCRwith subsequent LCR to identify the nucleotides at individual positionsis also a clearly cumbersome proposition for the clinical laboratory.

[0024] II. Direct Detection Technology

[0025] When a sufficient amount of a nucleic acid to be detected isavailable, there are advantages to detecting that sequence directly,instead of making more copies of that target, (e.g., as in PCR and LCR).Most notably, a method that does not amplify the signal exponentially ismore amenable to quantitative analysis. Even if the signal is enhancedby attaching multiple dyes to a single oligonucleotide, the correlationbetween the final signal intensity and amount of target is direct. Sucha system has an additional advantage that the products of the reactionwill not themselves promote further reaction, so contamination of labsurfaces by the products is not as much of a concern. Traditionalmethods of direct detection including Northern and Southern blotting andRNase protection assays usually require the use of radioactivity and arenot amenable to automation. Recently devised techniques have sought toeliminate the use of radioactivity and/or improve the sensitivity inautomatable formats. Two examples are the “Cycling Probe Reaction”(CPR), and “Branched DNA” (bDNA)

[0026] The cycling probe reaction (CPR) (Duck et al., BioTech., 9:142[1990]), uses a long chimeric oligonucleotide in which a central portionis made of RNA while the two termini are made of DNA. Hybridization ofthe probe to a target DNA and exposure to a thermostable RNase H causesthe RNA portion to be digested. This destabilizes the remaining DNAportions of the duplex, releasing the remainder of the probe from thetarget DNA and allowing another probe molecule to repeat the process.The signal, in the form of cleaved probe molecules, accumulates at alinear rate. While the repeating process increases the signal, the RNAportion of the oligonucleotide is vulnerable to RNases that may becarried through sample preparation.

[0027] Branched DNA (bDNA), described by Urdea et al., Gene 61:253-264(1987), involves oligonucleotides with branched structures that alloweach individual oligonucleotide to carry 35 to 40 labels (e.g., alkalinephosphatase enzymes). While this enhances the signal from ahybridization event, signal from non-specific binding is similarlyincreased.

[0028] While both of these methods have the advantages of directdetection discussed above, neither the CPR or bDNA methods can make useof the specificity allowed by the requirement of independent recognitionby two or more probe (oligonucleotide) sequences, as is common in thesignal amplification methods described in Section I. above. Therequirement that two oligonucleotides must hybridize to a target nucleicacid in order for a detectable signal to be generated confers an extrameasure of stringency on any detection assay. Requiring twooligonucleotides to bind to a target nucleic acid reduces the chancethat false “positive” results will be produced due to the non-specificbinding of a probe to the target. The further requirement that the twooligonucleotides must bind in a specific orientation relative to thetarget, as is required in PCR, where oligonucleotides must be oppositelybut appropriately oriented such that the DNA polymerase can bridge thegap between the two oligonucleotides in both directions, furtherenhances specificity of the detection reaction. However, it is wellknown to those in the art that even though PCR utilizes twooligonucleotide probes (termed primers) “non-specific” amplification(i.e., amplification of sequences not directed by the two primers used)is a common artifact. This is in part because the DNA polymerase used inPCR can accommodate very large distances, measured in nucleotides,between the oligonucleotides and thus there is a large window in whichnon-specific binding of an oligonucleotide can lead to exponentialamplification of inappropriate product. The LCR, in contrast, cannotproceed unless the oligonucleotides used are bound to the targetadjacent to each other and so the full benefit of the dualoligonucleotide hybridization is realized.

[0029] An ideal direct detection method would combine the advantages ofthe direct detection assays (e.g., easy quantification and minimal riskof carry-over contamination) with the specificity provided by a dualoligonucleotide hybridization assay.

SUMMARY OF THE INVENTION

[0030] The present invention relates to compositions and methods for thedetection and characterization of nucleic acid sequences and variationsin nucleic acid sequences. The present invention relates to methods forforming a nucleic acid cleavage structure on a target sequence andcleaving the nucleic acid cleavage structure in a site-specific manner.For example, in some embodiments, a 5′ nuclease activity of any of avariety of enzymes is used to cleave the target-dependent cleavagestructure, thereby indicating the presence of specific nucleic acidsequences or specific variations thereof.

[0031] The present invention provides structure-specific cleavage agents(e.g., nucleases) from a variety of sources, including mesophilic,psychrophilic, thermophilic, and hyperthermophilic organisms. Thepreferred structure-specific nucleases are thermostable. Thermostablestructure-specific nucleases are contemplated as particularly useful inthat they operate at temperatures where nucleic acid hybridization isextremely specific, allowing for allele-specific detection (includingsingle-base mismatches). In one embodiment, the thermostablestructure-specific nucleases are thermostable 5′ nucleases comprisingaltered polymerases derived from the native polymerases of Thermusspecies, including, but not limited to Thermus aquaticus, Thermusflavus, and Thermus thermophilus. However, the invention is not limitedto the use of thermostable 5′ nucleases. Thermostable structure-specificnucleases from the FEN-1, RAD2 and XPG class of nucleases are alsopreferred.

[0032] The present invention provides a method for detecting a targetsequence (e.g., a mutation, polymorphism, etc), comprising providing asample suspected of containing the target sequence; oligonucleotidescapable of forming an invasive cleavage structure in the presence of thetarget sequence; and an agent for detecting the presence of an invasivecleavage structure; and exposing the sample to the oligonucleotides andthe agent. In some embodiments, the method further comprises the step ofdetecting a complex comprising the agent and the invasive cleavagestructure (directly or indirectly). In some embodiments, the agentcomprises a cleavage agent. In some preferred embodiments, the exposingof the sample to the oligonucleotides and the agent comprises exposingthe sample to the oligonucleotides and the agent under conditionswherein an invasive cleavage structure is formed between the targetsequence and the oligonucleotides if the target sequence is present inthe sample, wherein the invasive cleavage structure is cleaved by thecleavage agent to form a cleavage product. In some embodiments, themethod further comprises the step of detecting the cleavage product. Insome embodiments, the target sequence comprises a first region and asecond region, the second region downstream of and contiguous to thefirst region, and wherein the oligonucleotides comprise first and secondoligonucleotides, wherein at least a portion of the firstoligonucleotide is completely complementary to the first portion of thetarget sequence and wherein the second oligonucleotide comprises a 3′portion and a 5′ portion, wherein the 5′ portion is completelycomplementary to the second portion of said target nucleic acid.

[0033] The present invention also provides a kit for detecting suchtarget sequences, said kit comprising oligonucleotides capable offorming an invasive cleavage structure in the presence of the targetsequence. In some embodiments, the kit further comprises an agent fordetecting the presence of an invasive cleavage structure (e.g., acleavage agent). In some embodiments, the oligonucleotides comprisefirst and second oligonucleotides, said first oligonucleotide comprisinga 5′ portion complementary to a first region of the target nucleic acidand said second oligonucleotide comprising a 3′ portion and a 5′portion, said 5′ portion complementary to a second region of the targetnucleic acid downstream of and contiguous to the first portion. In somepreferred embodiments, the target sequence comprises humancytomegalovirus viral DNA; sequence containing polymorphisms in thehuman apolipoprotein E gene (ApoE); sequence containing mutations in thehuman hemochromatosis (HH) gene; sequence containing mutations in humanMTHFR; sequence containing prothrombin 20210GA polymorphism; sequencecontaining HR-2 mutation in human factor V gene; sequence containingsingle nucleotide polymorphisms in human TNF-α gene, and sequencecontaining the Leiden mutation in human factor V gene. In some preferredembodiments, kits comprise oligonucleotides for detecting two or moretarget sequences. For example, information on two or more mutations mayprovide medically relevant information such that kits allowing detectionof the plurality of mutations would be desired (e.g., Factor V and HR-2detection). In some preferred embodiments kits are probed containing aprobe oligonucleotide comprising a sequence of SEQ ID NOs: 197, 198,199, 200, 208, 209, 211, 212, 217, 218, 223, 224, 229, 232, 236, 237,241, 242, or 244. In still other embodiments, kits provideoligonucleotide sets, the sets including one or more of theoligonucleotides: SEQ ID NOs: 195, 197, and 198 for ApoE detection; 196,199, and 200 for ApoE detection; 202, 208, and 209 for HH detection;203, 211, and 212 for HH detection; 216, 217, and 218 for MTHFRdetection; 222, 223, and 224 for prothrombin polymorphism detection;228, 229, 231, and 232 for HR-2 detection; 235, 236, and 237 for TNF-αdetection; 240, 241, and 242 for Factor V detection; and 243, 244, 246,and 247 for MRSA detection.

[0034] The present invention also provides methods for detecting thepresence of a target nucleic acid molecule by detecting non-targetcleavage products comprising providing: a cleavage agent; a source oftarget nucleic acid, the target nucleic acid comprising a first regionand a second region, the second region downstream of and contiguous tothe first region; a first oligonucleotide, wherein at least a portion ofthe first oligonucleotide is completely complementary to the firstportion of the target nucleic acid; and a second oligonucleotidecomprising a 3′ portion and a 5′ portion, wherein the 5′ portion iscompletely complementary to the second portion of the target nucleicacid; mixing the cleavage agent, the target nucleic acid, the firstoligonucleotide and the second oligonucleotide to create a reactionmixture under reaction conditions such that at least the portion of thefirst oligonucleotide is annealed to the first region of said targetnucleic acid and wherein at least the 5′ portion of the secondoligonucleotide is annealed to the second region of the target nucleicacid so as to create a cleavage structure, and wherein cleavage of thecleavage structure occurs to generate non-target cleavage product; anddetecting the cleavage of the cleavage structure.

[0035] The detection of the cleavage of the cleavage structure can becarried out in any manner. In some embodiments, the detection of thecleavage of the cleavage structure comprises detecting the non-targetcleavage product. In yet other embodiments, the detection of thecleavage of the cleavage structure comprises detection of fluorescence,mass, or fluorescence energy transfer. Other detection methods include,but are not limited to detection of radioactivity, luminescence,phosphorescence, fluorescence polarization, and charge. In someembodiments, detection is carried out by a method comprising providingthe non-target cleavage product; a composition comprising twosingle-stranded nucleic acids annealed so as to define a single-strandedportion of a protein binding region; and a protein; and exposing thenon-target cleavage product to the single-stranded portion of theprotein binding region under conditions such that the protein binds tothe protein binding region. In some embodiments, the protein comprises anucleic acid producing protein, wherein the nucleic acid producingprotein binds to the protein binding region and produces nucleic acid.In some embodiments, the protein binding region is a template-dependentRNA polymerase binding region (e.g., a T7 RNA polymerase bindingregion). In other embodiments, the detection is carried out by a methodcomprising providing the non-target cleavage product; a singlecontinuous strand of nucleic acid comprising a sequence defining asingle strand of an RNA polymerase binding region; a template-dependentDNA polymerase; and a template-dependent RNA polymerase; exposing thenon-target cleavage product to the RNA polymerase binding region underconditions such that the non-target cleavage product binds to a portionof the single strand of the RNA polymerase binding region to produce abound non-target cleavage product; exposing the bound non-targetcleavage product to the template-dependent DNA polymerase underconditions such that a double-stranded RNA polymerase binding region isproduced; and exposing the double-stranded RNA polymerase binding regionto the template-dependent RNA polymerase under conditions such that RNAtranscripts are produced. In some embodiments, the method furthercomprises the step of detecting the RNA transcripts. In someembodiments, the template-dependent RNA polymerase is T7 RNA polymerase.

[0036] The present invention is not limited by the nature of the 3′portion of the second oligonucleotide. In some preferred embodiments,the 3′ portion of the second oligonucleotide comprises a 3′ terminalnucleotide not complementary to the target nucleic acid. In someembodiments, the 3′ portion of the second oligonucleotide consists of asingle nucleotide not complementary to the target nucleic acid.

[0037] Any of the components of the method may be attached to a solidsupport. For example, in some embodiments, the first oligonucleotide isattached to a solid support. In other embodiments, the secondoligonucleotide is attached to a solid support.

[0038] The cleavage agent can be any agent that is capable of cleavinginvasive cleavage structures. In some preferred embodiments, thecleavage agent comprises a structure-specific nuclease. In particularlypreferred embodiments, the structure-specific nuclease comprises athermostable structure-specific nuclease (e.g., a thermostable 5′nuclease). Thermostable structure-specific nucleases include, but arenot limited to, those having an amino acid sequence homologous to aportion of the amino acid sequence of a thermostable DNA polymerasederived from a thermophilic organism (e.g., Thermus aquaticus, Thermusflavus, and Thermus thermophilus). In other embodiments, thethermostable structure-specific nucleases is from the FEN-1, RAD2 or XPGclass of nucleases, a chimerical structures containing one or moreportions of any of the above cleavage agents.

[0039] The method is not limited by the nature of the target nucleicacid. In some embodiments, the target nucleic acid is single stranded ordouble stranded DNA or RNA. In some embodiments, double stranded nucleicacid is rendered single stranded (e.g., by heat) prior to formation ofthe cleavage structure. In some embodiment, the source of target nucleicacid comprises a sample containing genomic DNA. Sample include, but arenot limited to, blood, saliva, cerebral spinal fluid, pleural fluid,milk, lymph, sputum and semen.

[0040] In some embodiments, the target nucleic acid comprises genomicDNA or mRNA. In other embodiments, the target nucleic acid comprisessynthetic DNA or RNA. In some preferred embodiments, synthetic DNA orRNA within a sample is created using a purified polymerase. In somepreferred embodiments, creation of synthetic DNA using a purifiedpolymerase comprises the use of PCR. In some preferred embodiments,creation of synthetic DNA comprises use of the methods and compositionsfor amplification using RNA-DNA composite primers (e.g., as disclosed inU.S. Pat. No. 6,251,639, herein incorporated by reference in itsentirety). In other preferred embodiments, creation of synthetic DNAusing a purified DNA polymerase suitable for use with the methods of thepresent invention comprises use of rolling circle amplification,(e.g.,as in U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502, hereinincorporated by reference in their entireties). In other preferredembodiments, creation of synthetic DNA comprises amplification usingnucleic acids comprising loop-forming sequences, e.g., as described inU.S. Pat. No. 6,410,278, herein incorporated by reference in itsentirety.

[0041] The present invention is not limited by the particular sequencesformed in the creation of synthetic DNA. In some embodiments, thesynthetic DNA sequence comprises at least a portion copied (e.g., bytemplate-directed synthesis using a template-dependent polymerase) froma natural nucleic acid (e.g., genomic DNA, mRNA, etc.). In otherembodiments, the creation of synthetic DNA comprises copying orreplication of a probe sequence, e.g., in response to the presence of atarget genomic DNA or mRNA sequence.

[0042] In some preferred embodiments, creation of synthetic DNAcomprises copying genomic DNA by priming from a plurality of sites on agenomic DNA sample. In some embodiments, priming from a plurality ofsites on a genomic DNA sample comprises using short (e.g., fewer thanabout 8 nucleotides) oligonucleotide primers. In other embodiments,priming from a plurality of sites on a genomic DNA comprises extensionof 3′ ends in nicked, double-stranded genomic DNA (i.e., where a 3′hydroxyl group has been made available for extension by breakage orcleavage of one strand of a double stranded region of DNA). Someexamples of making synthetic DNA using a purified polymerase on nickedgenomic DNAs, suitable for use with the methods and compositions of thepresent invention, are provided in U.S. Pat. Nos. 6,117,634, issued Sep.12, 2000, and 6,197,557, issued Mar. 6, 2001, and in PCT application WO98/39485, each incorporated by reference herein in their entireties forall purposes.

[0043] In some embodiments, the present invention provides methods fordetecting a target sequence, comprising: providing a) a samplecontaining synthetic DNA amplified from genomic DNA, said genomic DNAsuspected of containing said target sequence; b) oligonucleotidescapable of forming an invasive cleavage structure in the presence ofsaid target sequence; and c) exposing the sample to the oligonucleotidesand the agent. In some embodiments, the agent comprises a cleavageagent. In some particularly preferred embodiments, the method of theinvention further comprises the step of detecting said cleavage product.

[0044] In some particularly preferred embodiments, the genomic DNA isnicked double stranded genomic DNA, and the synthetic DNA is amplifiedby extension of 3′ ends in said nicked double-stranded genomic DNAsuspected of containing the target sequence.

[0045] In other particularly preferred embodiments, the synthetic DNAamplified from genomic DNA is amplified by extension of multiple primerson said genomic DNA suspected of containing said target sequence.

[0046] In some preferred embodiments, the exposing of the sample to theoligonucleotides and the agent comprises exposing the sample to theoligonucleotides and the agent under conditions wherein an invasivecleavage structure is formed between said target sequence and saidoligonucleotides if said target sequence is present in said sample,wherein said invasive cleavage structure is cleaved by said cleavageagent to form a cleavage product.

[0047] In some particularly preferred embodiments, the target sequencecomprises a first region and a second region, said second regiondownstream of and contiguous to said first region, and saidoligonucleotides comprise first and second oligonucleotides, saidwherein at least a portion of said first oligonucleotide is completelycomplementary to said first portion of said target sequence and whereinsaid second oligonucleotide comprises a 3′ portion and a 5′ portion,wherein said 5′ portion is completely complementary to said secondportion of said target nucleic acid.

[0048] In some embodiments, the target sequence is selected from thegroup consisting of human cytomegalovirus viral DNA; polymorphisms inhuman apolipoprotein E gene; mutations in human hemochromatosis gene;mutations in human MTHFR; prothrombin 20210GA polymorphism; HR-2mutation in human factor V gene; single nucleotide polymorphisms inhuman TNF-α gene, and Leiden mutation in human factor V gene.

[0049] In other embodiments, synthetic DNA suitable for use with themethods and compositions of the present invention is made using apurified polymerase on multiply-primed genomic DNA, as provided, e.g.,in U.S. Pat. Nos. 6,291,187, and 6,323,009, and in PCT applications WO01/88190 and WO 02/00934, each herein incorporated by reference in theirentireties for all purposes. In these embodiments, amplification of DNAsuch as genomic DNA is accomplished using a DNA polymerase, such as thehighly processive Φ29 polymerase (as described, e.g., in U.S. Pat. Nos.5,198,543 and 5,001,050, each herein incorporated by reference in theirentireties for all purposes) in combination with exonuclease-resistantrandom primers, such as hexamers.

[0050] In some embodiments, the present invention provides methods fordetecting a target sequence, comprising: providing a) a samplecontaining DNA amplified by extension of multiple primers on genomicDNA, said genomic DNA suspected of containing said target sequence; b)oligonucleotides capable of forming an invasive cleavage structure inthe presence of said target sequence; and c) exposing the sample to theoligonucleotides and the agent. In some embodiments, the agent comprisesa cleavage agent. In some preferred embodiments, said primers are randomprimers. In particularly preferred embodiments, said primers areexonuclease resistant. In some particularly preferred embodiments, themethod of the invention further comprises the step of detecting saidcleavage product.

[0051] In some preferred embodiments, the exposing of the sample to theoligonucleotides and the agent comprises exposing the sample to theoligonucleotides and the agent under conditions wherein an invasivecleavage structure is formed between said target sequence and saidoligonucleotides if said target sequence is present in said sample,wherein said invasive cleavage structure is cleaved by said cleavageagent to form a cleavage product.

[0052] In some preferred embodiments, the exposing of the sample to theoligonucleotides and the agent comprises exposing the sample to theoligonucleotides and the agent under conditions wherein an invasivecleavage structure is formed between said target sequence and saidoligonucleotides if said target sequence is present in said sample,wherein said invasive cleavage structure is cleaved by said cleavageagent to form a cleavage product.

[0053] In some particularly preferred embodiments, the target sequencecomprises a first region and a second region, said second regiondownstream of and contiguous to said first region, and saidoligonucleotides comprise first and second oligonucleotides, saidwherein at least a portion of said first oligonucleotide is completelycomplementary to said first portion of said target sequence and whereinsaid second oligonucleotide comprises a 3′ portion and a 5′ portion,wherein said 5′ portion is completely complementary to said secondportion of said target nucleic acid.

[0054] In some embodiments, the target sequence is selected from thegroup consisting of human cytomegalovirus viral DNA; polymorphisms inhuman apolipoprotein E gene; mutations in human hemochromatosis gene;mutations in human MTHFR; prothrombin 20210GA polymorphism; HR-2mutation in human factor V gene; single nucleotide polymorphisms inhuman TNF-α gene, and Leiden mutation in human factor V gene.

[0055] Synthetic DNAs produced using a purified polymerase on multiplyprimed or nicked genomic DNA also find use in detection assays thatinclude, but are not limited to, enzyme mismatch cleavage methods (e.g.,Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, hereinincorporated by reference in their entireties); polymerase chainreaction; branched hybridization methods (e.g., Chiron, U.S. Pat. Nos.5,849,481, 5,710,264, 5,124,246, and 5,624,802, herein incorporated byreference in their entireties); rolling circle replication (e.g., U.S.Pat. Nos. 6,210,884, 6,183,960 and 6,235,502, herein incorporated byreference in their entireties); NASBA (e.g., U.S. Pat. No. 5,409,818,herein incorporated by reference in its entirety); molecular beacontechnology (e.g., U.S. Pat. No. 6,150,097, herein incorporated byreference in its entirety); E-sensor technology (Motorola, U.S. Pat.Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, herein incorporatedby reference in their entireties); cycling probe technology (e.g., U.S.Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, herein incorporated byreference in their entireties); Dade Behring signal amplificationmethods (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230,5,882,867, and 5,792,614, herein incorporated by reference in theirentireties); ligase chain reaction (Barnay Proc. Natl. Acad. Sci USA 88,189-93 (1991)); and sandwich hybridization methods (e.g., U.S. Pat. No.5,288,609, herein incorporated by reference in its entirety).

[0056] In some embodiments, the reaction conditions for the methodcomprise providing a source of divalent cations. In some preferredembodiments, the divalent cation is selected from the group comprisingMn²⁺ and Mg²⁺ ions. In some embodiments, the reaction conditions for themethod comprise providing the first and the second oligonucleotides inconcentration excess compared to the target nucleic acid.

[0057] In some embodiments, the method further comprises providing athird oligonucleotide complementary to a third portion of said targetnucleic acid upstream of the first portion of the target nucleic acid,wherein the third oligonucleotide is mixed with the reaction mixture.

[0058] The present invention also provides a method for detecting thepresence of a target nucleic acid molecule by detecting non-targetcleavage products comprising providing: a cleavage agent; a source oftarget nucleic acid, the target nucleic acid comprising a first regionand a second region, the second region downstream of and contiguous tothe first region; a plurality of first oligonucleotides, wherein atleast a portion of the first oligonucleotides is completelycomplementary to the first portion of the target nucleic acid; a secondoligonucleotide comprising a 3′ portion and a 5′ portion, wherein said5′ portion is completely complementary to the second portion of thetarget nucleic acid; mixing the cleavage agent, the target nucleic acid,the plurality of first oligonucleotides and second oligonucleotide tocreate a reaction mixture under reaction conditions such that at leastthe portion of a first oligonucleotide is annealed to the first regionof the target nucleic acid and wherein at least the 5′ portion of thesecond oligonucleotide is annealed to the second region of the targetnucleic acid so as to create a cleavage structure, and wherein cleavageof the cleavage structure occurs to generate non-target cleavageproduct, wherein the conditions permit multiple cleavage structures toform and be cleaved from the target nucleic acid; and detecting thecleavage of said cleavage structures. In some embodiments, theconditions comprise isothermal conditions that permit the plurality offirst oligonucleotides to dissociate from the target nucleic acid. Whilethe present invention is limited by the number of cleavage structureformed on a particular target nucleic acid, in some preferredembodiments, two or more (3, 4, 5, . . . , 10, . . . , 10000, . . . ) ofthe plurality of first oligonucleotides form cleavage structures with aparticular target nucleic acid, wherein the cleavage structures arecleaved to produce the non-target cleavage products.

[0059] The present invention also provide methods where a cleavageproduct from the above methods is used in a further invasive cleavagereaction. For example, the present invention provides a methodcomprising providing a cleavage agent; a first target nucleic acid, thefirst target nucleic acid comprising a first region and a second region,the second region downstream of and contiguous to the first region; afirst oligonucleotide, wherein at least a portion of the firstoligonucleotide is completely complementary to the first portion of thefirst target nucleic acid; a second oligonucleotide comprising a 3′portion and a 5′ portion, wherein the 5′ portion is completelycomplementary to the second portion of the first target nucleic acid; asecond target nucleic acid, said second target nucleic acid comprising afirst region and a second region, the second region downstream of andcontiguous to the first region; and a third oligonucleotide, wherein atleast a portion of the third oligonucleotide is completely complementaryto the first portion of the second target nucleic acid; generating afirst cleavage structure wherein at least said portion of the firstoligonucleotide is annealed to the first region of the first targetnucleic acid and wherein at least the 5′ portion of the secondoligonucleotide is annealed to the second region of the first targetnucleic acid and wherein cleavage of the first cleavage structure occursvia the cleavage agent thereby cleaving the first oligonucleotide togenerate a fourth oligonucleotide, said fourth oligonucleotidecomprising a 3′ portion and a 5′ portion, wherein the 5′ portion iscompletely complementary to the second portion of the second targetnucleic acid; generating a second cleavage structure under conditionswherein at least said portion of the third oligonucleotide is annealedto the first region of the second target nucleic acid and wherein atleast the 5′ portion of the fourth oligonucleotide is annealed to thesecond region of the second target nucleic acid and wherein cleavage ofthe second cleavage structure occurs to generate a cleavage fragment;and detecting the cleavage of the second cleavage structure. In somepreferred embodiments, the 3′ portion of the fourth oligonucleotidecomprises a 3′ terminal nucleotide not complementary to the secondtarget nucleic acid. In some embodiments, the 3′ portion of the thirdoligonucleotide is covalently linked to the second target nucleic acid.In some embodiments, the second target nucleic acid further comprises a5′ region, wherein the 5′ region of the second target nucleic acid isthe third oligonucleotide. The present invention further provides kitscomprising: a cleavage agent; a first oligonucleotide comprising a 5′portion complementary to a first region of a target nucleic acid; and asecond oligonucleotide comprising a 3′ portion and a 5′ portion, said 5′portion complementary to a second region of the target nucleic aciddownstream of and contiguous to the first portion. In some embodiments,the 3′ portion of the second oligonucleotide comprises a 3′ terminalnucleotide not complementary to the target nucleic acid. In preferredembodiments, the 3′ portion of the second oligonucleotide consists of asingle nucleotide not complementary to the target nucleic acid. In someembodiments, the kit further comprises a solid support. For example, insome embodiments, the first and/or second oligonucleotide is attached tosaid solid support. In some embodiments, the kit further comprises abuffer solution. In some preferred embodiments, the buffer solutioncomprises a source of divalent cations (e.g., Mn²⁺ and/or Mg²⁺ ions). Insome specific embodiments, the kit further comprises a thirdoligonucleotide complementary to a third portion of the target nucleicacid upstream of the first portion of the first target nucleic acid. Inyet other embodiments, the kit further comprises a target nucleic acid.In some embodiments, the kit further comprises a second target nucleicacid. In yet other embodiments, the kit further comprises a thirdoligonucleotide comprising a 5′ portion complementary to a first regionof the second target nucleic acid. In some specific embodiments, the 3′portion of the third oligonucleotide is covalently linked to the secondtarget nucleic acid. In other specific embodiments, the second targetnucleic acid further comprises a 5′ portion, wherein the 5′ portion ofthe second target nucleic acid is the third oligonucleotide. In stillother embodiments, the kit further comprises an ARRESTOR molecule (e.g.,ARRESTOR oligonucleotide).

[0060] The present invention further provides a composition comprising acleavage structure, the cleavage structure comprising: a) a targetnucleic acid, the target nucleic acid having a first region, a secondregion, a third region and a fourth region, wherein the first region islocated adjacent to and downstream from the second region, the secondregion is located adjacent to and downstream from the third region andthe third region is located adjacent to and downstream from the fourthregion; b) a first oligonucleotide complementary to the fourth region ofthe target nucleic acid; c) a second oligonucleotide having a 5′ portionand a 3′ portion wherein the 5′ portion of the second oligonucleotidecontains a sequence complementary to the second region of the targetnucleic acid and wherein the 3′ portion of the second oligonucleotidecontains a sequence complementary to the third region of the targetnucleic acid; and d) a third oligonucleotide having a 5′ portion and a3′ portion wherein the 5′ portion of the third oligonucleotide containsa sequence complementary to the first region of the target nucleic acidand wherein the 3′ portion of the third oligonucleotide contains asequence complementary to the second region of the target nucleic acid.

[0061] The present invention is not limited by the length of the fourregions of the target nucleic acid. In one embodiment, the first regionof the target nucleic acid has a length of 11 to 50 nucleotides. Inanother embodiment, the second region of the target nucleic acid has alength of one to three nucleotides. In another embodiment, the thirdregion of the target nucleic acid has a length of six to ninenucleotides. In yet another embodiment, the fourth region of the targetnucleic acid has a length of 6 to 50 nucleotides.

[0062] The invention is not limited by the nature or composition of theof the first, second, third and fourth oligonucleotides; theseoligonucleotides may comprise DNA, RNA, PNA and combinations thereof aswell as comprise modified nucleotides, universal bases, adducts, etc.Further, one or more of the first, second, third and the fourtholigonucleotides may contain a dideoxynucleotide at the 3′ terminus.

[0063] In one preferred embodiment, the target nucleic acid is notcompletely complementary to at least one of the first, the second, thethird and the fourth oligonucleotides. In a particularly preferredembodiment, the target nucleic acid is not completely complementary tothe second oligonucleotide.

[0064] As noted above, the present invention contemplates the use ofstructure-specific nucleases in detection methods. In one embodiment,the present invention provides a method of detecting the presence of atarget nucleic acid molecule by detecting non-target cleavage productscomprising: a) providing: i) a cleavage means, ii) a source of targetnucleic acid, the target nucleic acid having a first region, a secondregion, a third region and a fourth region, wherein the first region islocated adjacent to and downstream from the second region, the secondregion is located adjacent to and downstream from the third region andthe third region is located adjacent to and downstream from the fourthregion; iii) a first oligonucleotide complementary to the fourth regionof the target nucleic acid; iv) a second oligonucleotide having a 5′portion and a 3′ portion wherein the 5′ portion of the secondoligonucleotide contains a sequence complementary to the second regionof the target nucleic acid and wherein the 3′ portion of the secondoligonucleotide contains a sequence complementary to the third region ofthe target nucleic acid; iv) a third oligonucleotide having a 5′ and a3′ portion wherein the 5′ portion of the third oligonucleotide containsa sequence complementary to the first region of the target nucleic acidand wherein the 3′ portion of the third oligonucleotide contains asequence complementary to the second region of the target nucleic acid;b) mixing the cleavage means, the target nucleic acid, the firstoligonucleotide, the second oligonucleotide and the thirdoligonucleotide to create a reaction mixture under reaction conditionssuch that the first oligonucleotide is annealed to the fourth region ofthe target nucleic acid and wherein at least the 3′ portion of thesecond oligonucleotide is annealed to the target nucleic acid andwherein at least the 5′ portion of the third oligonucleotide is annealedto the target nucleic acid so as to create a cleavage structure andwherein cleavage of the cleavage structure occurs to generate non-targetcleavage products, each non-target cleavage product having a 3′-hydroxylgroup; and c) detecting the non-target cleavage products.

[0065] The invention is not limited by the nature of the target nucleicacid. In one embodiment, the target nucleic acid comprisessingle-stranded DNA. In another embodiment, the target nucleic acidcomprises double-stranded DNA and prior to step c), the reaction mixtureis treated such that the double-stranded DNA is rendered substantiallysingle-stranded. In another embodiment, the target nucleic acidcomprises RNA and the first and second oligonucleotides comprise DNA.

[0066] The invention is not limited by the nature of the cleavage means.In one embodiment, the cleavage means is a structure-specific nuclease;particularly preferred structure-specific nucleases are thermostablestructure-specific nucleases. In one preferred embodiment, thethermostable structure-specific nuclease is encoded by a DNA sequenceselected from the group consisting of SEQ ID NOS: 1-3, 9, 10, 12, 21,30, 31, 101, 106, 110, 114, 129, 131, 132, 137, 140, 141, 142, 143, 144,145, 147, 150, 151, 153, 155, 156, 157, 158, 161, 163, 178, 180, and182.

[0067] In another preferred embodiment, the thermostablestructure-specific nuclease is a nuclease from the FEN-1/RAD2/XPG classof nucleases. In another preferred embodiment the thermostable structurespecific nuclease is a chimerical nuclease.

[0068] In an alternative preferred embodiment, the detection of thenon-target cleavage products comprises electrophoretic separation of theproducts of the reaction followed by visualization of the separatednon-target cleavage products.

[0069] In another preferred embodiment, one or more of the first,second, and third oligonucleotides contain a dideoxynucleotide at the 3′terminus. When dideoxynucleotide-containing oligonucleotides areemployed, the detection of the non-target cleavage products preferablycomprises: a) incubating the non-target cleavage products with atemplate-independent polymerase and at least one labeled nucleosidetriphosphate under conditions such that at least one labeled nucleotideis added to the 3′-hydroxyl group of the non-target cleavage products togenerate labeled non-target cleavage products; and b) detecting thepresence of the labeled non-target cleavage products. The invention isnot limited by the nature of the template-independent polymeraseemployed; in one embodiment, the template-independent polymerase isselected from the group consisting of terminal deoxynucleotidyltransferase (TdT) and poly A polymerase. When TdT or polyA polymeraseare employed in the detection step, the second oligonucleotide maycontain a 5′ end label, the 5′ end label being a different label thanthe label present upon the labeled nucleoside triphosphate. Theinvention is not limited by the nature of the 5′ end label; a widevariety of suitable 5′ end labels are known to the art and includebiotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3amidite, Cy5 amidite and digoxigenin.

[0070] In another embodiment, detecting the non-target cleavage productscomprises: a) incubating the non-target cleavage products with atemplate-independent polymerase and at least one nucleoside triphosphateunder conditions such that at least one nucleotide is added to the3′-hydroxyl group of the non-target cleavage products to generate tailednon-target cleavage products; and b) detecting the presence of thetailed non-target cleavage products. The invention is not limited by thenature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerase are employed in the detectionstep, the second oligonucleotide may contain a 5′ end label. Theinvention is not limited by the nature of the 5′ end label; a widevariety of suitable 5′ end labels are known to the art and includebiotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3amidite, Cy5 amidite and digoxigenin.

[0071] In a preferred embodiment, the reaction conditions compriseproviding a source of divalent cations; particularly preferred divalentcations are Mn²⁺ and Mg²⁺ ions.

[0072] The present invention further provides a method of detecting thepresence of a target nucleic acid molecule by detecting non-targetcleavage products comprising: a) providing: i) a cleavage means, ii) asource of target nucleic acid, the target nucleic acid having a firstregion, a second region and a third region, wherein the first region islocated adjacent to and downstream from the second region and whereinthe second region is located adjacent to and downstream from the thirdregion; iii) a first oligonucleotide having a 5′ and a 3′ portionwherein the 5′ portion of the first oligonucleotide contains a sequencecomplementary to the second region of the target nucleic acid andwherein the 3′ portion of the first oligonucleotide contains a sequencecomplementary to the third region of the target nucleic acid; iv) asecond oligonucleotide having a length between eleven to fifteennucleotides and further having a 5′ and a 3′ portion wherein the 5′portion of the second oligonucleotide contains a sequence complementaryto the first region of the target nucleic acid and wherein the 3′portion of the second oligonucleotide contains a sequence complementaryto the second region of the target nucleic acid; b) mixing the cleavagemeans, the target nucleic acid, the first oligonucleotide and the secondoligonucleotide to create a reaction mixture under reaction conditionssuch that at least the 3′ portion of the first oligonucleotide isannealed to the target nucleic acid and wherein at least the 5′ portionof the second oligonucleotide is annealed to the target nucleic acid soas to create a cleavage structure and wherein cleavage of the cleavagestructure occurs to generate non-target cleavage products, eachnon-target cleavage product having a 3′-hydroxyl group; and c) detectingthe non-target cleavage products. In a preferred embodiment the cleavagemeans is a structure-specific nuclease, preferably a thermostablestructure-specific nuclease.

[0073] The invention is not limited by the length of the various regionsof the target nucleic acid. In a preferred embodiment, the second regionof the target nucleic acid has a length between one to five nucleotides.In another preferred embodiment, one or more of the first and the secondoligonucleotides contain a dideoxynucleotide at the 3′ terminus. Whendideoxynucleotide-containing oligonucleotides are employed, thedetection of the non-target cleavage products preferably comprises: a)incubating the non-target cleavage products with a template-independentpolymerase and at least one labeled nucleoside triphosphate underconditions such that at least one labeled nucleotide is added to the3′-hydroxyl group of the non-target cleavage products to generatelabeled non-target cleavage products; and b) detecting the presence ofthe labeled non-target cleavage products. The invention is not limitedby the nature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerase is employed in the detectionstep, the second oligonucleotide may contain a 5′ end label, the 5′ endlabel being a different label than the label present upon the labelednucleoside triphosphate. The invention is not limited by the nature ofthe 5′ end label; a wide variety of suitable 5′ end labels are known tothe art and include biotin, fluorescein, tetrachlorofluorescein,hexachlorofluorescein, Cy3 amidite, Cy5 amidite and digoxigenin.

[0074] In another embodiment, detecting the non-target cleavage productscomprises: a) incubating the non-target cleavage products with atemplate-independent polymerase and at least one nucleoside triphosphateunder conditions such that at least one nucleotide is added to the3′-hydroxyl group of the non-target cleavage products to generate tailednon-target cleavage products; and b) detecting the presence of thetailed non-target cleavage products. The invention is not limited by thenature of the template-independent polymerase employed; in oneembodiment, the template-independent polymerase is selected from thegroup consisting of terminal deoxynucleotidyl transferase (TdT) and polyA polymerase. When TdT or polyA polymerase are employed in the detectionstep, the second oligonucleotide may contain a 5′ end label. Theinvention is not limited by the nature of the 5′ end label; a widevariety of suitable 5′ end labels are known to the art and includebiotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3amidite, Cy5 amidite and digoxigenin.

[0075] The novel detection methods of the invention may be employed forthe detection of target DNAs and RNAs including, but not limited to,target DNAs and RNAs comprising wild type and mutant alleles of genes,including genes from humans or other animals that are or may beassociated with disease or cancer. In addition, the methods of theinvention may be used for the detection of and/or identification ofstrains of microorganisms, including bacteria, fungi, protozoa, ciliatesand viruses (and in particular for the detection and identification ofRNA viruses, such as HCV).

[0076] The present invention further provides improved enzymaticcleavage means. In one embodiment, the present invention provides athermostable structure-specific nuclease having an amino acid sequenceselected from the group consisting of SEQ ID NOS: 102, 107, 130, 132,179, 181, 183, 184, 185, 186, 187, and 188. In another embodiment, thenuclease is encoded by a DNA sequence selected from the group consistingof SEQ ID NOS: 101, 106, 129 131, 178, 180, and 182.

[0077] The present invention also provides a recombinant DNA vectorcomprising DNA having a nucleotide sequence encoding astructure-specific nuclease, the nucleotide sequence selected from thegroup consisting of SEQ ID NOS: 101, 106, 129 131, 137, 140, 141, 142,143, 144, 145, 147, 150, 151, 153, 155, 156, 157, 158, 161, 163, 178,180, and 182. In a preferred embodiment, the invention provides a hostcell transformed with a recombinant DNA vector comprising DNA having anucleotide sequence encoding a structure-specific nuclease, thenucleotide sequence selected from the group consisting of SEQ ID NOS:101, 106, 129, 131, 178, 180, and 182. The invention is not limited bythe nature of the host cell employed. The art is well aware ofexpression vectors suitable for the expression of nucleotide sequencesencoding structure-specific nucleases which can be expressed in avariety of prokaryotic and eukaryotic host cells. In a preferredembodiment, the host cell is an Escherichia coli cell.

[0078] The present invention provides purified FEN-1 endonucleases. Inone embodiment, the present invention provides Pyrococcus woesei FEN-1endonuclease. In a preferred embodiment, the purified Pyrococcus woeseiFEN-1 endonuclease has a molecular weight of about 38.7 kilodaltons (themolecular weight may be conveniently estimated using SDS-PAGE asdescribed in Ex. 28).

[0079] The present invention further provides an isolatedoligonucleotide encoding a Pyrococcus woesei FEN-1 endonuclease, theoligonucleotide having a region capable of hybridizing to anoligonucleotide sequence selected from the group consisting of SEQ IDNOS: 116-119. In a preferred embodiment, the oligonucleotide encodingthe purified Pyrococcus woesei FEN-1 endonuclease is operably linked toa heterologous promoter. The present invention is not limited by thenature of the heterologous promoter employed; in a preferred embodiment,the heterologous promoter is an inducible promoter (the promoter chosenwill depend upon the host cell chosen for expression as is known in theart). The invention is not limited by the nature of the induciblepromoter employed. Preferred inducible promoters include the −P_(L)promoter, the tac promoter, the trp promoter and the trc promoter.

[0080] In another preferred embodiment, the invention provides arecombinant DNA vector comprising an isolated oligonucleotide encoding aPyrococcus woesei (Pwo) FEN-1 endonuclease, the oligonucleotide having aregion capable of hybridizing to an oligonucleotide sequence selectedfrom the group consisting of SEQ ID NOS: 116-119. Host cells transformedwith these recombinant vectors are also provided. In a preferredembodiment, the invention provides a host cell transformed with arecombinant DNA vector comprising DNA having a region capable ofhybridizing to an oligonucleotide sequence selected from the groupconsisting of SEQ ID NOS: 116-119; these vectors may further comprise aheterologous promoter operably linked to the Pwo FEN-1-encodingpolynucleotides. The invention is not limited by the nature of the hostcell employed. The art is well aware of expression vectors suitable forthe expression of Pwo FEN-1-encoding polynucleotides which can beexpressed in a variety of prokaryotic and eukaryotic host cells. In apreferred embodiment, the host cell is an Escherichia coli cell.

[0081] In yet another embodiment, the invention provides an isolatedoligonucleotide comprising a gene encoding a Pyrococcus woesei FEN-1endonuclease having a molecular weight of about 38.7 kilodaltons. Inanother embodiment, the encoding a Pyrococcus woesei FEN-1 endonucleaseis operably linked to a heterologous promoter. The present invention isnot limited by the nature of the heterologous promoter employed; in apreferred embodiment, the heterologous promoter is an inducible promoter(the promoter chosen will depend upon the host cell chosen forexpression as is known in the art). The invention is not limited by thenature of the inducible promoter employed. Preferred inducible promoterinclude the −P_(L) promoter, the tac promoter, the trp promoter and thetrc promoter.

[0082] The invention further provides recombinant DNA vectors comprisingDNA having a nucleotide sequence encoding FEN-1 endonucleases. In onepreferred embodiment, the present invention provides a Pyrococcus woeseiFEN-1 endonuclease having a molecular weight of about 38.7 kilodaltons.Still further, a host cell transformed with a recombinant DNA vectorcomprising DNA having a nucleotide sequence encoding FEN-1 endonuclease.In a preferred embodiment, the host cell is transformed with arecombinant DNA vector comprising DNA having a nucleotide sequenceencoding a Pyrococcus woesei FEN-1 endonuclease having a molecularweight of about 38.7 kilodaltons is provided. The invention is notlimited by the nature of the host cell employed. The art is well awareof expression vectors suitable for the expression of Pwo FEN-1-encodingpolynucleotides which can be expressed in a variety of prokaryotic andeukaryotic host cells. In a preferred embodiment, the host cell is anEscherichia coli cell.

[0083] Thus, the present invention provides multiple purified FEN-1endonucleases, both purified native forms of the endonucleases, as wellas recombinant endonucleases. In preferred embodiments, the purifiedFEN-1 endonucleases are obtained from archaebacterial or eubacterialorganisms. In particularly preferred embodiments, the FEN-1endonucleases are obtained from organisms selected from the groupconsisting of Archaeoglobus fulgidus and Methanobacteriumthermoautotrophicum. In a preferred embodiment, the purified FEN-1endonucleases have molecular weights of about 39 kilodaltons (themolecular weight may be conveniently estimated using SDS-PAGE asdescribed in Ex. 28).

[0084] The present invention further provides isolated oligonucleotidesencoding Archaeoglobus fulgidus and Methanobacterium thermoautotrophicumFEN-1 endonucleases, the oligonucleotides each having a region capableof hybridizing to at least a portion of an oligonucleotide sequence,wherein the oligonucleotide sequence is selected from the groupconsisting of SEQ ID NOS: 170, 171, 172, and 173. In some preferredembodiment, the oligonucleotides encoding the Archaeoglobus fulgidus andMethanobacterium thermoautotrophicum FEN-1 endonucleases are operablylinked to heterologous promoters. However, it is not intended that thepresent invention be limited by the nature of the heterologous promoteremployed. It is contemplated that the promoter chosen will depend uponthe host cell chosen for expression as is known in the art. In somepreferred embodiments, the heterologous promoter is an induciblepromoter. The invention is not limited by the nature of the induciblepromoter employed. Preferred inducible promoters include the −P_(L)promoter, the tac promoter, the trp promoter and the trc promoter.

[0085] In another preferred embodiment, the invention providesrecombinant DNA vectors comprising isolated oligonucleotides encodingArchaeoglobus fulgidus or Methanobacterium thermoautotrophicum FEN-1endonucleases, each oligonucleotides having a region capable ofhybridizing to at least a portion of an oligonucleotide sequence,wherein the oligonucleotide sequence is selected from the groupconsisting of SEQ ID NOS: 170, 171, 172, and 173. The present inventionfurther provides host cells transformed with these recombinant vectors.In a preferred embodiment, the invention provides a host celltransformed with a recombinant DNA vector comprising DNA having a regioncapable of hybridizing to at least a portion of an oligonucleotidesequence, wherein the oligonucleotide sequence is selected from thegroup consisting of SEQ ID NOS: 170, 171, 172 and 173. In someembodiments, these vectors may further comprise a heterologous promoteroperably linked to the FEN-1-encoding polynucleotides. The invention isnot limited by the nature of the host cell employed. The art is wellaware of expression vectors suitable for the expression ofFEN-1-encoding polynucleotides which can be expressed in a variety ofprokaryotic and eukaryotic host cells. In a preferred embodiment, thehost cell is an Escherichia coli cell.

[0086] The present invention further provides chimericstructure-specific nucleases. In one embodiment, the present inventionprovides chimeric endonucleases comprising amino acid portions derivedfrom the endonucleases selected from the group of FEN-1, XPG and RADhomologs. In a preferred embodiment, the chimeric endonucleases compriseamino acid portions derived from the FEN-1 endonucleases selected fromthe group of Pyrococcus furiosus, Methanococcus jannaschi, Pyrococcuswoesei, Archaeoglobus fulgidus and Methanobacterium thermoautotrophicum.In a more preferred embodiment, the chimeric FEN-1 endonucleases havemolecular weights of about 39 kilodaltons (the molecular weight may beconveniently estimated using SDS-PAGE as described in Ex. 28).

[0087] The present invention further provides isolated oligonucleotidesencoding chimeric endonucleases. In one embodiment, the oligonucleotidesencoding the chimeric endonucleases comprise nucleic acid sequencesderived from the genes selected from the group of FEN-1, XPG and RADhomologs. In a preferred embodiment the oligonucleotides encoding thechimeric endonucleases comprise nucleic acid sequences derived from thegenes encoding the FEN-1 endonucleases selected from the group ofPyrococcus furiosus, Methanococcus jannaschi, Pyrococcus woesei,Archaeoglobus fulgidus and Methanobacterium thermoautotrophicum. In aparticularly preferred embodiment, the genes for the chimericendonucleases are operably linked to heterologous promoters. The presentinvention is not limited by the nature of the heterologous promoteremployed. It is contemplated that the promoter chosen will depend uponthe host cell selected for expression, as is known in the art. Inpreferred embodiments, the heterologous promoter is an induciblepromoter. The invention is not limited by the nature of the induciblepromoter employed. Preferred inducible promoter include the −P_(L)promoter, the tac promoter, the trp promoter and the trc promoter.

[0088] In another preferred embodiment, the invention providesrecombinant DNA vectors comprising isolated oligonucleotides encodingthe chimeric endonucleases described above. In one embodiment, therecombinant DNA vectors comprise isolated oligonucleotides encodingnucleic acid sequences derived from the genes selected from the group ofFEN-1, XPG and RAD homologs. In a preferred embodiment, the recombinantDNA vectors comprise isolated oligonucleotides encoding the chimericendonucleases comprising nucleic acid sequences derived from the genesencoding the FEN-1 endonucleases selected from the group of Pyrococcusfuriosus, Methanococcus jannaschi, Pyrococcus woesei, Archaeoglobusfulgidus and Methanobacterium thermoautotrophicum. These vectors mayfurther comprise a heterologous promoter operably linked to the chimericnuclease-encoding polynucleotides.

[0089] Host cells transformed with these recombinant vectors are alsoprovided. The invention is not limited by the nature of the host cellemployed. The art is well aware of expression vectors suitable for theexpression of FEN-1-encoding polynucleotides which can be expressed in avariety of prokaryotic and eukaryotic host cells. In a preferredembodiment, the host cell is an Escherichia coli cell.

[0090] The present invention further provides mixtures comprising afirst structure-specific nuclease, wherein the first nuclease consistsof a purified FEN-1 endonuclease and a second structure-specificnuclease. In preferred embodiments, the second structure-specificnuclease of the mixture is selected from the group consisting Pyrococcuswoesei FEN-1 endonuclease, Pyrococcus furiosus FEN-1, Methanococcusjannaschii FEN-1 endonuclease, Methanobacterium thermoautotrophicumFEN-1 endonuclease, Archaeoglobus fulgidus FEN-1, and chimerical FEN-1endonucleases. In alternative embodiments, the purified FEN-1endonuclease of the mixture is selected from the group consistingPyrococcus woesei FEN-1 endonuclease, Pyrococcus furiosus FEN-1endonuclease, Methanococcus jannaschii FEN-1 endonuclease,Methanobacterium thermoautotrophicum FEN-1 endonuclease, Archaeoglobusfulgidus FEN-1, and chimerical FEN-1 endonucleases. In yet otherpreferred embodiments of the mixture, the second nuclease is a 5′nuclease derived from a thermostable DNA polymerase altered in aminoacid sequence such that it exhibits reduced DNA synthetic activity fromthat of the wild-type DNA polymerase but retains substantially the same5′ nuclease activity of the wild-type DNA polymerase. In some preferredembodiments of the mixture, the second nuclease is selected from thegroup consisting of the Cleavase® BN enzyme, Thermus aquaticus DNApolymerase, Thermus thermophilus DNA polymerase, Escherichia coli ExoIII, Saccharomyces cerevisiae Rad1/Rad10 complex.

[0091] The present invention also provides methods for treating nucleicacid, comprising: a) providing a purified FEN-1 endonuclease; and anucleic acid substrate; b) treating the nucleic acid substrate underconditions such that the substrate forms one or more cleavagestructures; and c) reacting the endonuclease with the cleavagestructures so that one or more cleavage products are produced. In someembodiments, the purified FEN-1 endonuclease is selected from the groupconsisting Pyrococcus woesei FEN-1 endonuclease, Pyrococcus furiosusFEN-1 endonuclease, Methanococcus jannaschii FEN-1 endonuclease,Methanobacterium thermoautotrophicum FEN-1 endonuclease, Archaeoglobusfulgidus FEN-1, and chimerical FEN-1 endonucleases. In otherembodiments, the method further comprises providing a structure-specificnuclease derived from a thermostable DNA polymerase altered in aminoacid sequence such that it exhibits reduced DNA synthetic activity fromthat of the wild-type DNA polymerase but retains substantially the same5′ nuclease activity of the wild-type DNA polymerase.

[0092] In alternative embodiments of the methods, a portion of the aminoacid sequence of the second nuclease is homologous to a portion of theamino acid sequence of a thermostable DNA polymerase derived from aeubacterial thermophile of the genus Thermus. In yet other embodiments,the thermophile is selected from the group consisting of Thermusaquaticus, Thermus flavus and Thermus thermophilus. In some alternativeembodiments, the structure-specific nuclease is selected from the groupconsisting of the Cleavase® BN enzyme, Thermus aquaticus DNA polymerase,Thermus thermophilus DNA polymerase, Escherichia coli Exo III,Saccharomyces cerevisiae Rad1/Rad10 complex. In some preferredembodiments, the structure-specific nuclease is the Cleavase® BNnuclease. In yet other embodiments, the nucleic acid of step (a) issubstantially single-stranded. In further embodiments, the nucleic acidis selected from the group consisting of RNA and DNA. In yet furtherembodiments, the nucleic acid of step (a) is double stranded.

[0093] In other embodiments of the methods, the treating of step (b)comprises: rendering the double-stranded nucleic acid substantiallysingle-stranded; and exposing the single-stranded nucleic acid toconditions such that the single-stranded nucleic acid has secondarystructure. In some preferred embodiments, the double stranded nucleicacid is rendered substantially single-stranded by the use of increasedtemperature. In alternative preferred embodiments, the method furthercomprises the step of detecting the one or more cleavage products.

[0094] The present invention also provides methods for treating nucleicacid, comprising: a) providing: a first structure-specific nucleaseconsisting of a purified FEN-1 endonuclease in a solution containingmanganese; and a nucleic acid substrate; b) treating the nucleic acidsubstrate with increased temperature such that the substrate issubstantially single-stranded; c) reducing the temperature underconditions such that the single-stranded substrate forms one or morecleavage structures; d) reacting the cleavage means with the cleavagestructures so that one or more cleavage products are produced; and e)detecting the one or more cleavage products. In some embodiments of themethods, the purified FEN-1 endonuclease is selected from the groupconsisting Pyrococcus woesei FEN-1 endonuclease, Pyrococcus furiosusFEN-1 endonuclease, Methanococcus jannaschii FEN-1 endonuclease,Methanobacterium thermoautotrophicum FEN-1 endonuclease, Archaeoglobusfulgidus FEN-1, and chimerical FEN-1 endonucleases. In alternativeembodiments, the methods further comprise providing a secondstructure-specific nuclease. In some preferred embodiments, the secondnuclease is selected from the group consisting of the Cleavase® BNenzyme, Thermus aquaticus DNA polymerase, Thermus thermophilus DNApolymerase, Escherichia coli Exo III, and the Saccharomyces cerevisiaeRad1/Rad10 complex. In yet other preferred embodiments, the secondnuclease is a 5′ nuclease derived from a thermostable DNA polymerasealtered in amino acid sequence such that it exhibits reduced DNAsynthetic activity from that of the wild-type DNA polymerase but retainssubstantially the same 5′ nuclease activity of the wild-type DNApolymerase. In yet other embodiments, the nucleic acid is selected fromthe group consisting of RNA and DNA. In further embodiments, the nucleicacid of step (a) is double stranded.

[0095] The present invention also provides nucleic acid treatment kits,comprising: a) a composition comprising at least one purified FEN-1endonuclease; and b) a solution containing manganese. In someembodiments of the kits, the purified FEN-1 endonuclease is selectedfrom the group consisting Pyrococcus woesei FEN-1 endonuclease,Pyrococcus furiosus FEN-1 endonuclease, Methanococcus jannaschii FEN-1endonuclease, Methanobacterium thermoautotrophicum FEN-1 endonuclease,Archaeoglobus fulgidus FEN-1, and chimerical FEN-1 endonucleases. Inother embodiments, the kits further comprise at least one secondstructure-specific nuclease. In some preferred embodiments, the secondnuclease is a 5′ nuclease derived from a thermostable DNA polymerasealtered in amino acid sequence such that it exhibits reduced DNAsynthetic activity from that of the wild-type DNA polymerase but retainssubstantially the same 5′ nuclease activity of the wild-type DNApolymerase. In yet other embodiments of the kits, the portion of theamino acid sequence of the second nuclease is homologous to a portion ofthe amino acid sequence of a thermostable DNA polymerase derived from aeubacterial thermophile of the genus Thermus. In further embodiments,the thermophile is selected from the group consisting of Thermusaquaticus, Thermus flavus and Thermus thermophilus. In yet otherpreferred embodiments, the kits further comprise reagents for detectingthe cleavage products.

[0096] The present invention also provides methods for improving themethods and enzymes disclosed herein. For example, the present inventionprovides methods of improving enzymes for any intended purpose (e.g.,use in cleavage reactions, amplification reactions, binding reactions,or any other use) comprising the step of providing an enzyme disclosedherein and modifying the enzyme (e.g., altering the amino acid sequence,adding or subtracting sequence, adding post-translational modifications,adding any other component whether biological or not, or any othermodification). Likewise, the present invention provides methods forimproving the methods disclosed herein comprising, conducting the methodsteps with one or more changes (e.g., change in a composition providedin the method, change in the order of the steps, or addition orsubtraction of steps).

[0097] Definitions

[0098] To facilitate an understanding of the present invention, a numberof terms and phrases are defined below:

[0099] As used herein, the terms “complementary” or “complementarity”are used in reference to polynucleotides (i.e., a sequence ofnucleotides such as an oligonucleotide or a target nucleic acid) relatedby the base-pairing rules. For example, for the sequence “5′-A-G-T-3′,”is complementary to the sequence “3′-T-C-A-5′.” Complementarity may be“partial,” in which only some of the nucleic acids bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods which depend upon binding between nucleicacids. Either term may also be used in reference to individualnucleotides, especially within the context of polynucleotides. Forexample, a particular nucleotide within an oligonucleotide may be notedfor its complementarity, or lack thereof, to a nucleotide within anothernucleic acid strand, in contrast or comparison to the complementaritybetween the rest of the oligonucleotide and the nucleic acid strand.

[0100] The term “homology” and “homologous” refers to a degree ofidentity. There may be partial homology or complete homology. Apartially homologous sequence is one that is less than 100% identical toanother sequence.

[0101] As used herein, the term “hybridization” is used in reference tothe pairing of complementary nucleic acids. Hybridization and thestrength of hybridization (i.e., the strength of the association betweenthe nucleic acids) is influenced by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, and the T_(m) of the formed hybrid. “Hybridization” methodsinvolve the annealing of one nucleic acid to another, complementarynucleic acid, i.e., a nucleic acid having a complementary nucleotidesequence. The ability of two polymers of nucleic acid containingcomplementary sequences to find each other and anneal through basepairing interaction is a well-recognized phenomenon. The initialobservations of the “hybridization” process by Marmur and Lane, Proc.Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad.Sci. USA 46:461 (1960) have been followed by the refinement of thisprocess into an essential tool of modem biology.

[0102] With regard to complementarity, it is important for somediagnostic applications to determine whether the hybridizationrepresents complete or partial complementarity. For example, where it isdesired to detect simply the presence or absence of pathogen DNA (suchas from a virus, bacterium, fungi, mycoplasma, protozoan) it is onlyimportant that the hybridization method ensures hybridization when therelevant sequence is present; conditions can be selected where bothpartially complementary probes and completely complementary probes willhybridize. Other diagnostic applications, however, may require that thehybridization method distinguish between partial and completecomplementarity. It may be of interest to detect genetic polymorphisms.For example, human hemoglobin is composed, in part, of four polypeptidechains. Two of these chains are identical chains of 141 amino acids(alpha chains) and two of these chains are identical chains of 146 aminoacids (beta chains). The gene encoding the beta chain is known toexhibit polymorphism. The normal allele encodes a beta chain havingglutamic acid at the sixth position. The mutant allele encodes a betachain having valine at the sixth position. This difference in aminoacids has a profound (most profound when the individual is homozygousfor the mutant allele) physiological impact known clinically as sicklecell anemia. It is well known that the genetic basis of the amino acidchange involves a single base difference between the normal allele DNAsequence and the mutant allele DNA sequence.

[0103] The complement of a nucleic acid sequence as used herein refersto an oligonucleotide which, when aligned with the nucleic acid sequencesuch that the 5′ end of one sequence is paired with the 3′ end of theother, is in “antiparallel association.” Certain bases not commonlyfound in natural nucleic acids may be included in the nucleic acids ofthe present invention and include, for example, inosine and7-deazaguanine. Complementarity need not be perfect; stable duplexes maycontain mismatched base pairs or unmatched bases. Those skilled in theart of nucleic acid technology can determine duplex stabilityempirically considering a number of variables including, for example,the length of the oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

[0104] As used herein, the term “T_(m)” is used in reference to the“melting temperature.” The melting temperature is the temperature atwhich a population of double-stranded nucleic acid molecules becomeshalf dissociated into single strands. Several equations for calculatingthe T_(m) of nucleic acids are well known in the art. As indicated bystandard references, a simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl (see e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985).Other references (e.g., Allawi, H. T. & SantaLucia, J., Jr.Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry36, 10581-94 (1997) include more sophisticated computations which takestructural and environmental, as well as sequence characteristics intoaccount for the calculation of T_(m).

[0105] As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “weak” or “low”stringency are often required when it is desired that nucleic acidswhich are not completely complementary to one another be hybridized orannealed together.

[0106] “High stringency conditions” when used in reference to nucleicacid hybridization comprise conditions equivalent to binding orhybridization at 42 C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmonsperm DNA followed by washing in a solution comprising 0.1×SSPE, 1.0%SDS at 42 C. when a probe of about 500 nucleotides in length isemployed. “Medium stringency conditions” when used in reference tonucleic acid hybridization comprise conditions equivalent to binding orhybridization at 42 C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmonsperm DNA followed by washing in a solution comprising 1.0×SSPE, 1.0%SDS at 42 C. when a probe of about 500 nucleotides in length isemployed.

[0107] “Low stringency conditions” comprise conditions equivalent tobinding or hybridization at 42 C. in a solution consisting of 5×SSPE(43.8 g/l NaCl, 6.9 g/l NaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to7.4 with NaOH), 0.1% SDS, 5×Denhardt's reagent [50×Denhardt's containsper 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V;Sigma)] and 100 g/ml denatured salmon sperm DNA followed by washing in asolution comprising 5×SSPE, 0.1 % SDS at 42 C. when a probe of about 500nucleotides in length is employed.

[0108] The term “gene” refers to a DNA sequence that comprises controland coding sequences necessary for the production of an RNA having anon-coding function (e.g., a ribosomal or transfer RNA), a polypeptideor a precursor. The RNA or polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence so long as thedesired activity or function is retained.

[0109] The term “wild-type” refers to a gene or a gene product that hasthe characteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified”, “mutant” or “polymorphic” refers to a gene or gene productwhich displays modifications in sequence and or functional properties(i.e., altered characteristics) when compared to the wild-type gene orgene product. It is noted that naturally-occurring mutants can beisolated; these are identified by the fact that they have alteredcharacteristics when compared to the wild-type gene or gene product.

[0110] The term “recombinant DNA vector” as used herein refers to DNAsequences containing a desired heterologous sequence. For example,although the term is not limited to the use of expressed sequences orsequences that encode an expression product, in some embodiments, theheterologous sequence is a coding sequence and appropriate DNA sequencesnecessary for either the replication of the coding sequence in a hostorganism, or the expression of the operably linked coding sequence in aparticular host organism. DNA sequences necessary for expression inprokaryotes include a promoter, optionally an operator sequence, aribosome binding site and possibly other sequences. Eukaryotic cells areknown to utilize promoters, polyadenlyation signals and enhancers.

[0111] The term “LTR” as used herein refers to the long terminal repeatfound at each end of a provirus (i.e., the integrated form of aretrovirus). The LTR contains numerous regulatory signals includingtranscriptional control elements, polyadenylation signals and sequencesneeded for replication and integration of the viral genome. The viralLTR is divided into three regions called U3, R and U5.

[0112] The U3 region contains the enhancer and promoter elements. The U5region contains the polyadenylation signals. The R (repeat) regionseparates the U3 and U5 regions and transcribed sequences of the Rregion appear at both the 5′ and 3′ ends of the viral RNA.

[0113] The term “oligonucleotide” as used herein is defined as amolecule comprising two or more deoxyribonucleotides or ribonucleotides,preferably at least 5 nucleotides, more preferably at least about 10-15nucleotides and more preferably at least about 15 to 30 nucleotides. Theexact size will depend on many factors, which in turn depend on theultimate function or use of the oligonucleotide. The oligonucleotide maybe generated in any manner, including chemical synthesis, DNAreplication, reverse transcription, PCR, or a combination thereof.

[0114] Because mononucleotides are reacted to make oligonucleotides in amanner such that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction.

[0115] When two different, non-overlapping oligonucleotides anneal todifferent regions of the same linear complementary nucleic acidsequence, and the 3′ end of one oligonucleotide points towards the 5′end of the other, the former may be called the “upstream”oligonucleotide and the latter the “downstream” oligonucleotide.Similarly, when two overlapping oligonucleotides are hybridized to thesame linear complementary nucleic acid sequence, with the firstoligonucleotide positioned such that its 5′ end is upstream of the 5′end of the second oligonucleotide, and the 3′ end of the firstoligonucleotide is upstream of the 3′ end of the second oligonucleotide,the first oligonucleotide may be called the “upstream” oligonucleotideand the second oligonucleotide may be called the “downstream”oligonucleotide.

[0116] The term “primer” refers to an oligonucleotide that is capable ofacting as a point of initiation of synthesis when placed underconditions in which primer extension is initiated. An oligonucleotide“primer” may occur naturally, as in a purified restriction digest or maybe produced synthetically.

[0117] A primer is selected to be “substantially” complementary to astrand of specific sequence of the template. A primer must besufficiently complementary to hybridize with a template strand forprimer elongation to occur. A primer sequence need not reflect the exactsequence of the template. For example, a non-complementary nucleotidefragment may be attached to the 5′ end of the primer, with the remainderof the primer sequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

[0118] The term “label” as used herein refers to any atom or moleculethat can be used to provide a detectable (preferably quantifiable)signal, and that can be attached to a nucleic acid or protein. Labelsmay provide signals detectable by fluorescence, radioactivity,colorimetry, gravimetry, X-ray diffraction or absorption, magnetism,enzymatic activity, and the like. A label may be a charged moiety(positive or negative charge) or alternatively, may be charge neutral.Labels can include or consist of nucleic acid or protein sequence, solong as the sequence comprising the label is detectable.

[0119] The term “cleavage structure” as used herein, refers to astructure that is formed by the interaction of at least one probeoligonucleotide and a target nucleic acid, forming a structurecomprising a duplex, the resulting structure being cleavable by acleavage means, including but not limited to an enzyme. The cleavagestructure is a substrate for specific cleavage by the cleavage means incontrast to a nucleic acid molecule that is a substrate for non-specificcleavage by agents such as phosphodiesterases which cleave nucleic acidmolecules without regard to secondary structure (i.e., no formation of aduplexed structure is required).

[0120] The term “folded cleavage structure” as used herein, refers to aregion of a single-stranded nucleic acid substrate containing secondarystructure, the region being cleavable by an enzymatic cleavage means.The cleavage structure is a substrate for specific cleavage by thecleavage means in contrast to a nucleic acid molecule which is asubstrate for non-specific cleavage by agents such as phosphodiesteraseswhich cleave nucleic acid molecules without regard to secondarystructure (i.e., no folding of the substrate is required).

[0121] As used herein, the term “folded target” refers to a nucleic acidstrand that contains at least one region of secondary structure (i.e.,at least one double stranded region and at least one single-strandedregion within a single strand of the nucleic acid). A folded target maycomprise regions of tertiary structure in addition to regions ofsecondary structure.

[0122] The term “cleavage means” or “cleavage agent” as used hereinrefers to any means that is capable of cleaving a cleavage structure,including but not limited to enzymes. The cleavage means may includenative DNAPs having 5′ nuclease activity (e.g., Taq DNA polymerase, E.coli DNA polymerase I) and, more specifically, modified DNAPs having 5′nuclease but lacking synthetic activity. “Structure-specific nucleases”or “structure-specific enzymes” are enzymes that recognize specificsecondary structures in a nucleic molecule and cleave these structures.The cleavage means of the invention cleave a nucleic acid molecule inresponse to the formation of cleavage structures; it is not necessarythat the cleavage means cleave the cleavage structure at any particularlocation within the cleavage structure.

[0123] The cleavage means is not restricted to enzymes having solely 5′nuclease activity. The cleavage means may include nuclease activityprovided from a variety of sources including the Cleavase enzymes, theFEN-1 endonucleases (including RAD2 and XPG proteins), Taq DNApolymerase and E. coli DNA polymerase I.

[0124] The term “thermostable” when used in reference to an enzyme, suchas a 5′ nuclease, indicates that the enzyme is functional or active(i.e., can perform catalysis) at an elevated temperature, i.e., at about55° C. or higher.

[0125] The term “cleavage products” as used herein, refers to productsgenerated by the reaction of a cleavage means with a cleavage structure(i.e., the treatment of a cleavage structure with a cleavage means).

[0126] The term “target nucleic acid” refers to a nucleic acid moleculecontaining a sequence that has at least partial complementarity with atleast a probe oligonucleotide and may also have at least partialcomplementarity with an INVADER oligonucleotide. The target nucleic acidmay comprise single- or double-stranded DNA or RNA.

[0127] The term “probe oligonucleotide” refers to an oligonucleotidethat interacts with a target nucleic acid to form a cleavage structurein the presence or absence of an INVADER oligonucleotide. When annealedto the target nucleic acid, the probe oligonucleotide and target form acleavage structure and cleavage occurs within the probe oligonucleotide.

[0128] The term “non-target cleavage product” refers to a product of acleavage reaction that is not derived from the target nucleic acid. Asdiscussed above, in the methods of the present invention, cleavage ofthe cleavage structure generally occurs within the probeoligonucleotide. The fragments of the probe oligonucleotide generated bythis target nucleic acid-dependent cleavage are “non-target cleavageproducts.”

[0129] The term “INVADER oligonucleotide” refers to an oligonucleotidethat hybridizes to a target nucleic acid at a location near the regionof hybridization between a probe and the target nucleic acid, whereinthe INVADER oligonucleotide comprises a portion (e.g., a chemicalmoiety, or nucleotide—whether complementary to that target or not) thatoverlaps with the region of hybridization between the probe and target.In some embodiments, the INVADER oligonucleotide contains sequences atits 3′ end that are substantially the same as sequences located at the5′ end of a probe oligonucleotide.

[0130] The term “substantially single-stranded” when used in referenceto a nucleic acid substrate means that the substrate molecule existsprimarily as a single strand of nucleic acid in contrast to adouble-stranded substrate which exists as two strands of nucleic acidwhich are held together by inter-strand base pairing interactions.

[0131] The term “sequence variation” as used herein refers todifferences in nucleic acid sequence between two nucleic acids. Forexample, a wild-type structural gene and a mutant form of this wild-typestructural gene may vary in sequence by the presence of single basesubstitutions and/or deletions or insertions of one or more nucleotides.These two forms of the structural gene are said to vary in sequence fromone another. A second mutant form of the structural gene may exist. Thissecond mutant form is said to vary in sequence from both the wild-typegene and the first mutant form of the gene.

[0132] The term “liberating” as used herein refers to the release of anucleic acid fragment from a larger nucleic acid fragment, such as anoligonucleotide, by the action of, for example, a 5′ nuclease such thatthe released fragment is no longer covalently attached to the remainderof the oligonucleotide.

[0133] The term “K_(m)” as used herein refers to the Michaelis-Mentenconstant for an enzyme and is defined as the concentration of thespecific substrate at which a given enzyme yields one-half its maximumvelocity in an enzyme catalyzed reaction.

[0134] The term “nucleotide analog” as used herein refers to modified ornon-naturally occurring nucleotides such as 7-deaza purines (i.e.,7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogsand comprise modified forms of deoxyribonucleotides as well asribonucleotides.

[0135] The term “polymorphic locus” is a locus present in a populationthat shows variation between members of the population (e.g.., the mostcommon allele has a frequency of less than 0.95). In contrast, a“monomorphic locus” is a genetic locus at little or no variations seenbetween members of the population (generally taken to be a locus atwhich the most common allele exceeds a frequency of 0.95 in the genepool of the population).

[0136] The term “microorganism” as used herein means an organism toosmall to be observed with the unaided eye and includes, but is notlimited to bacteria, virus, protozoans, fungi, and ciliates.

[0137] The term “microbial gene sequences” refers to gene sequencesderived from a microorganism.

[0138] The term “bacteria” refers to any bacterial species includingeubacterial and archaebacterial species.

[0139] The term “virus” refers to obligate, ultramicroscopic,intracellular parasites incapable of autonomous replication (i.e.,replication requires the use of the host cell's machinery).

[0140] The term “multi-drug resistant” or multiple-drug resistant”refers to a microorganism which is resistant to more than one of theantibiotics or antimicrobial agents used in the treatment of saidmicroorganism.

[0141] The term “sample” in the present specification and claims is usedin its broadest sense. On the one hand it is meant to include a specimenor culture (e.g., microbiological cultures). On the other hand, it ismeant to include both biological and environmental samples. A sample mayinclude a specimen of synthetic origin.

[0142] Biological samples may be animal, including human, fluid, solid(e.g., stool) or tissue, as well as liquid and solid food and feedproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Biological samples may be obtained from all ofthe various families of domestic animals, as well as feral or wildanimals, including, but not limited to, such animals as ungulates, bear,fish, lagamorphs, rodents, etc.

[0143] Environmental samples include environmental material such assurface matter, soil, water and industrial samples, as well as samplesobtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items. These examplesare not to be construed as limiting the sample types applicable to thepresent invention.

[0144] The term “source of target nucleic acid” refers to any samplethat contains nucleic acids (RNA or DNA). Particularly preferred sourcesof target nucleic acids are biological samples including, but notlimited to blood, saliva, cerebral spinal fluid, pleural fluid, milk,lymph, sputum and semen.

[0145] An oligonucleotide is said to be present in “excess” relative toanother oligonucleotide (or target nucleic acid sequence) if thatoligonucleotide is present at a higher molar concentration that theother oligonucleotide (or target nucleic acid sequence). When anoligonucleotide such as a probe oligonucleotide is present in a cleavagereaction in excess relative to the concentration of the complementarytarget nucleic acid sequence, the reaction may be used to indicate theamount of the target nucleic acid present. Typically, when present inexcess, the probe oligonucleotide will be present at least a 100-foldmolar excess; typically at least 1 pmole of each probe oligonucleotidewould be used when the target nucleic acid sequence was present at about10 fmoles or less.

[0146] A sample “suspected of containing” a first and a second targetnucleic acid may contain either, both or neither target nucleic acidmolecule.

[0147] The term “charge-balanced” oligonucleotide refers to anoligonucleotide (the input oligonucleotide in a reaction) that has beenmodified such that the modified oligonucleotide bears a charge, suchthat when the modified oligonucleotide is either cleaved (i.e.,shortened) or elongated, a resulting product bears a charge differentfrom the input oligonucleotide (the “charge-unbalanced” oligonucleotide)thereby permitting separation of the input and reacted oligonucleotideson the basis of charge. The term “charge-balanced” does not imply thatthe modified or balanced oligonucleotide has a net neutral charge(although this can be the case). Charge-balancing refers to the designand modification of an oligonucleotide such that a specific reactionproduct generated from this input oligonucleotide can be separated onthe basis of charge from the input oligonucleotide.

[0148] For example, in an INVADER oligonucleotide-directed cleavageassay in which the probe oligonucleotide bears the sequence:5′TTCTTTTCACCAGCGAGACGGG 3′ (i.e., SEQ ID NO: 61 without the modifiedbases) and cleavage of the probe occurs between the second and thirdresidues, one possible charge-balanced version of this oligonucleotidewould be: 5′Cy3-AminoT-Amino-TCTTTTCACCAGCGAGAC GGG 3′. This modifiedoligonucleotide bears a net negative charge. After cleavage, thefollowing oligonucleotides are generated: 5′Cy3-AminoT-Amino-T 3′ and5′CTTTTCACCAGCGAGACGGG 3′ (residues 3-22 of SEQ ID NO: 61).5′Cy3-AminoT-Amino-T 3′ bears a detectable moiety (thepositively-charged Cy3 dye) and two amino-modified bases. Theamino-modified bases and the Cy3 dye contribute positive charges inexcess of the negative charges contributed by the phosphate groups andthus the 5′Cy3-AminoT-Amino-T 3′ oligonucleotide has a net positivecharge. The other, longer cleavage fragment, like the input probe, bearsa net negative charge. Because the 5′Cy3-AminoT-Amino-T 3′ fragment isseparable on the basis of charge from the input probe (thecharge-balanced oligonucleotide), it is referred to as acharge-unbalanced oligonucleotide. The longer cleavage product cannot beseparated on the basis of charge from the input oligonucleotide as botholigonucleotides bear a net negative charge; thus, the longer cleavageproduct is not a charge-unbalanced oligonucleotide.

[0149] The term “net neutral charge” when used in reference to anoligonucleotide, including modified oligonucleotides, indicates that thesum of the charges present (i.e., R—NH3+ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction or separation conditions is essentially zero.An oligonucleotide having a net neutral charge would not migrate in anelectrical field.

[0150] The term “net positive charge” when used in reference to anoligonucleotide, including modified oligonucleotides, indicates that thesum of the charges present (i.e., R—NH3+ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction conditions is +1 or greater. Anoligonucleotide having a net positive charge would migrate toward thenegative electrode in an electrical field.

[0151] The term “net negative charge” when used in reference to anoligonucleotide, including modified oligonucleotides, indicates that thesum of the charges present (i.e., R—NH3+ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction conditions is −1 or lower. An oligonucleotidehaving a net negative charge would migrate toward the positive electrodein an electrical field.

[0152] The term “polymerization means” or “polymerization agent” refersto any agent capable of facilitating the addition of nucleosidetriphosphates to an oligonucleotide. Preferred polymerization meanscomprise DNA and RNA polymerases.

[0153] The term “ligation means” or “ligation agent” refers to any agentcapable of facilitating the ligation (i.e., the formation of aphosphodiester bond between a 3′-OH and a 5′ P located at the termini oftwo strands of nucleic acid). Preferred ligation means comprise DNAligases and RNA ligases.

[0154] The term “reactant” is used herein in its broadest sense. Thereactant can comprise, for example, an enzymatic reactant, a chemicalreactant or light (e.g., ultraviolet light, particularly shortwavelength ultraviolet light is known to break oligonucleotide chains).Any agent capable of reacting with an oligonucleotide to either shorten(i.e., cleave) or elongate the oligonucleotide is encompassed within theterm “reactant.”

[0155] The term “adduct” is used herein in its broadest sense toindicate any compound or element that can be added to anoligonucleotide. An adduct may be charged (positively or negatively) ormay be charge-neutral. An adduct may be added to the oligonucleotide viacovalent or non-covalent linkages. Examples of adducts include, but arenot limited to, indodicarbocyanine dye amidites, amino-substitutednucleotides, ethidium bromide, ethidium homodimer,(1,3-propanediamino)propidium, (diethylenetriamino)propidium, thiazoleorange, (N-N′-tetramethyl-1,3-propanediamino)propyl thiazole orange,(N-N′-tetramethyl-1,2-ethanediamino)propyl thiazole orange, thiazoleorange-thiazole orange homodimer (TOTO), thiazole orange-thiazole blueheterodimer (TOTAB), thiazole orange-ethidium heterodimer 1 (TOED1),thiazole orange-ethidium heterodimer 2 (TOED2) and fluorescein-ethidiumheterodimer (FED), psoralens, biotin, streptavidin, avidin, etc.

[0156] Where a first oligonucleotide is complementary to a region of atarget nucleic acid and a second oligonucleotide has complementary tothe same region (or a portion of this region) a “region of overlap”exists along the target nucleic acid. The degree of overlap will varydepending upon the nature of the complementarity (see, e.g., region “X”in FIGS. 29 and 67 and the accompanying discussions).

[0157] As used herein, the term “purified” or “to purify” refers to theremoval of contaminants from a sample. For example, recombinant Cleavasenucleases are expressed in bacterial host cells and the nucleases arepurified by the removal of host cell proteins; the percent of theserecombinant nucleases is thereby increased in the sample.

[0158] The term “recombinant DNA molecule” as used herein refers to aDNA molecule that comprises of segments of DNA joined together by meansof molecular biological techniques.

[0159] The term “recombinant protein” or “recombinant polypeptide” asused herein refers to a protein molecule that is expressed from arecombinant DNA molecule.

[0160] As used herein the term “portion” when in reference to a protein(as in “a portion of a given protein”) refers to fragments of thatprotein. The fragments may range in size from four amino acid residuesto the entire amino acid sequence minus one amino acid (e.g., 4, 5, 6, .. ., n−1).

[0161] The term “nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin which may besingle or double stranded, and represent the sense or antisense strand.Similarly, “amino acid sequence” as used herein refers to peptide orprotein sequence.

[0162] The term “peptide nucleic acid” (“PNA”) as used herein refers toa molecule comprising bases or base analogs such as would be found innatural nucleic acid, but attached to a peptide backbone rather than thesugar-phosphate backbone typical of nucleic acids. The attachment of thebases to the peptide is such as to allow the bases to base pair withcomplementary bases of nucleic acid in a manner similar to that of anoligonucleotide. These small molecules, also designated anti geneagents, stop transcript elongation by binding to their complementarystrand of nucleic acid (Nielsen, et al. Anticancer Drug Des. 8:53 63[1993]).

[0163] As used herein, the terms “purified” or “substantially purified”refer to molecules, either nucleic or amino acid sequences, that areremoved from their natural environment, isolated or separated, and areat least 60% free, preferably 75% free, and most preferably 90% freefrom other components with which they are naturally associated. An“isolated polynucleotide” or “isolated oligonucleotide” is therefore asubstantially purified polynucleotide.

[0164] An isolated oligonucleotide (or polynucleotide) encoding aPyrococcus woesei (Pwo) FEN-1 endonuclease having a region capable ofhybridizing to SEQ ID NO: 116 is an oligonucleotide containing sequencesencoding at least the amino-terminal portion of Pwo FEN-1 endonuclease.An isolated oligonucleotide (or polynucleotide) encoding a Pwo FEN-1endonuclease having a region capable of hybridizing to SEQ ID NO: 117 isan oligonucleotide containing sequences encoding at least thecarboxy-terminal portion of Pwo FEN-1 endonuclease. An isolatedoligonucleotide (or polynucleotide) encoding a Pwo FEN-1 endonucleasehaving a region capable of hybridizing to SEQ ID NOS: 118 and 119 is anoligonucleotide containing sequences encoding at least portions of PwoFEN-1 endonuclease protein located internal to either the amino orcarboxy-termini of the Pwo FEN-1 endonuclease protein.

[0165] As used herein, the term “fusion protein” refers to a chimericprotein containing the protein of interest (e.g., Cleavase BN/thrombinnuclease and portions or fragments thereof) joined to an exogenousprotein fragment (the fusion partner which consists of a non CleavaseBN/thrombin nuclease protein). The fusion partner may enhance solubilityof recombinant chimeric protein (e.g., the Cleavase BN/thrombinnuclease) as expressed in a host cell, may provide an affinity tag(e.g., a his-tag) to allow purification of the recombinant fusionprotein from the host cell or culture supernatant, or both. If desired,the fusion protein may be removed from the protein of interest (e.g.,Cleavase BN/thrombin nuclease or fragments thereof) by a variety ofenzymatic or chemical means known to the art.

[0166] As used herein, the terms “chimeric protein” and “chimericalprotein” refer to a single protein molecule that comprises amino acidsequences portions derived from two or more parent proteins. Theseparent molecules may be from similar proteins from genetically distinctorigins, different proteins from a single organism, or differentproteins from different organisms. By way of example but not by way oflimitation, a chimeric structure-specific nuclease of the presentinvention may contain a mixture of amino acid sequences that have beenderived from FEN-1 genes from two or more of the organisms having suchgenes, combined to form a non-naturally occurring nuclease. The term“chimerical” as used herein is not intended to convey any particularproportion of contribution from the naturally occurring genes, nor limitthe manner in which the portions are combined. Any chimericstructure-specific nuclease constructs having cleavage activity asdetermined by the testing methods described herein are improved cleavageagents within the scope of the present invention.

[0167] The term “continuous strand of nucleic acid” as used herein ismeans a strand of nucleic acid that has a continuous, covalently linked,backbone structure, without nicks or other disruptions. The dispositionof the base portion of each nucleotide, whether base-paired,single-stranded or mismatched, is not an element in the definition of acontinuous strand. The backbone of the continuous strand is not limitedto the ribose-phosphate or deoxyribose-phosphate compositions that arefound in naturally occurring, unmodified nucleic acids. A nucleic acidof the present invention may comprise modifications in the structure ofthe backbone, including but not limited to phosphorothioate residues,phosphonate residues, 2′ substituted ribose residues (e.g., 2′-O-methylribose) and alternative sugar (e.g., arabinose) containing residues.

[0168] The term “continuous duplex” as used herein refers to a region ofdouble stranded nucleic acid in which there is no disruption in theprogression of basepairs within the duplex (i.e., the base pairs alongthe duplex are not distorted to accommodate a gap, bulge or mismatchwith the confines of the region of continuous duplex). As used hereinthe term refers only to the arrangement of the basepairs within theduplex, without implication of continuity in the backbone portion of thenucleic acid strand. Duplex nucleic acids with uninterruptedbasepairing, but with nicks in one or both strands are within thedefinition of a continuous duplex.

[0169] The term “duplex” refers to the state of nucleic acids in whichthe base portions of the nucleotides on one strand are bound throughhydrogen bonding the their complementary bases arrayed on a secondstrand. The condition of being in a duplex form reflects on the state ofthe bases of a nucleic acid. By virtue of base pairing, the strands ofnucleic acid also generally assume the tertiary structure of a doublehelix, having a major and a minor groove. The assumption of the helicalform is implicit in the act of becoming duplexed.

[0170] The term “duplex dependent protein binding” refers to the bindingof proteins to nucleic acid that is dependent on the nucleic acid beingin a duplex, or helical form.

[0171] The term “duplex dependent protein binding sites or regions” asused herein refers to discrete regions or sequences within a nucleicacid that are bound with particular affinity by specificduplex-dependent nucleic acid binding proteins. This is in contrast tothe generalized duplex-dependent binding of proteins that are notsite-specific, such as the histone proteins that bind chromatin withlittle reference to specific sequences or sites.

[0172] The term “protein binding region” as used herein refers to anucleic acid region identified by a sequence or structure as binding toa particular protein or class of proteins. It is within the scope ofthis definition to include those regions that contain sufficient geneticinformation to allow identifications of the region by comparison toknown sequences, but which might not have the requisite structure foractual binding (e.g., a single strand of a duplex-depending nucleic acidbinding protein site). As used herein “protein binding region” excludesrestriction endonuclease binding regions.

[0173] The term “complete double stranded protein binding region” asused herein refers to the minimum region of continuous duplex requiredto allow binding or other activity of a duplex-dependent protein. Thisdefinition is intended to encompass the observation that some duplexdependent nucleic acid binding proteins can interact with full activitywith regions of duplex that may be shorter than a canonical proteinbinding region as observed in one or the other of the two singlestrands. In other words, one or more nucleotides in the region may beallowed to remain unpaired without suppressing binding. As used here in,the term “complete double stranded binding region” refers to the minimumsequence that will accommodate the binding function. Because some suchregions can tolerate non-duplex sequences in multiple places, althoughnot necessarily simultaneously, a single protein binding region mighthave several shorter sub-regions that, when duplexed, will be fullycompetent for protein binding.

[0174] The term “template” refers to a strand of nucleic acid on which acomplementary copy is built from nucleoside triphosphates through theactivity of a template-dependent nucleic acid polymerase. Within aduplex the template strand is, by convention, depicted and described asthe “bottom” strand. Similarly, the non-template strand is oftendepicted and described as the “top” strand.

[0175] The term “template-dependent RNA polymerase” refers to a nucleicacid polymerase that creates new RNA strands through the copying of atemplate strand as described above and which does not synthesize RNA inthe absence of a template. This is in contrast to the activity of thetemplate-independent nucleic acid polymerases that synthesize or extendnucleic acids without reference to a template, such as terminaldeoxynucleotidyl transferase, or Poly A polymerase.

[0176] The term “ARRESTOR” refers to an agent added to or included in aninvasive cleavage reaction in order to stop one or more reactioncomponents from participating in a subsequent action or reaction. Thismay be done by sequestering or inactivating some reaction component(e.g., by binding or base-pairing a nucleic acid component, or bybinding to a protein component). The term “ARRESTOR oligonucleotide”refers to an oligonucleotide included in an invasive cleavage reactionin order to stop or arrest one or more aspects of any reaction (e.g.,the first reaction and/or any subsequent reactions or actions; it is notintended that the ARRESTOR oligonucleotide be limited to any particularreaction or reaction step). This may be done by sequestering somereaction component (e.g., base-pairing to another nucleic acid, orbinding to a protein component). However, it is not intended that theterm be so limited as to just situations in which a reaction componentis sequestered.

DESCRIPTION OF THE DRAWINGS

[0177]FIG. 1 is a comparison of the nucleotide structure of the DNAPgenes isolated from Thermus aquaticus (SEQ ID NO: 1), Thermus flavus(SEQ ID NO: 2) and Thermus thermophilus (SEQ ID NO: 3); the consensussequence (SEQ ID NO: 7) is shown at the top of each row.

[0178]FIG. 2 is a comparison of the amino acid sequence of the DNAPisolated from Thermus aquaticus (SEQ ID NO: 4), Thermus flavus (SEQ IDNO: 5), and Thermus thermophilus (SEQ ID NO: 6); the consensus sequence(SEQ ID NO: 8) is shown at the top of each row.

[0179] FIGS. 3A-G are a set of diagrams of wild-type andsynthesis-deficient DNAPTaq genes.

[0180]FIG. 4A depicts the wild-type Thermus flavus polymerase gene.

[0181]FIG. 4B depicts a synthesis-deficient Thermus flavus polymerasegene.

[0182]FIG. 5 depicts a structure which cannot be amplified usingDNAPTaq; this Figure shows SEQ ID NO: 17 (primer) and SEQ ID NO: 15(hairpin).

[0183]FIG. 6 is a ethidium bromide-stained gel demonstrating attempts toamplify a bifurcated duplex using either DNAPTaq or DNAPStf (i.e., theStoffel fragment of DNAPTaq).

[0184]FIG. 7 is an autoradiogram of a gel analyzing the cleavage of abifurcated duplex by DNAPTaq and lack of cleavage by DNAPStf.

[0185] FIGS. 8A-B are a set of autoradiograms of gels analyzing cleavageor lack of cleavage upon addition of different reaction components andchange of incubation temperature during attempts to cleave a bifurcatedduplex with DNAPTaq.

[0186] FIGS. 9A-B are an autoradiogram displaying timed cleavagereactions, with and without primer.

[0187] FIGS. 10A-B are a set of autoradiograms of gels demonstratingattempts to cleave a bifurcated duplex (with and without primer) withvarious DNAPs.

[0188]FIG. 11A shows the substrate and oligonucleotides (19-12 [SEQ IDNO: 18] and 30-12 [SEQ ID NO: 19]) used to test the specific cleavage ofsubstrate DNAs targeted by pilot oligonucleotides.

[0189]FIG. 11B shows an autoradiogram of a gel showing the results ofcleavage reactions using the substrates and oligonucleotides shown FIG.12A.

[0190]FIG. 12A shows the substrate and oligonucleotide (30-0 [SEQ ID NO:20]) used to test the specific cleavage of a substrate RNA targeted by apilot oligonucleotide.

[0191]FIG. 12B shows an autoradiogram of a gel showing the results of acleavage reaction using the substrate and oligonucleotide shown in FIG.13A.

[0192]FIG. 13 is a diagram of vector pTTQ18.

[0193]FIG. 14 is a diagram of vector pET-3c.

[0194] FIGS. 15A-E depicts a set of molecules which are suitablesubstrates for cleavage by the 5′ nuclease activity of DNAPs (SEQ IDNOS: 15 and 17 are depicted in FIG. 15E).

[0195]FIG. 16 is an autoradiogram of a gel showing the results of acleavage reaction run with synthesis-deficient DNAPs.

[0196]FIG. 17 is an autoradiogram of a PEI chromatogram resolving theproducts of an assay for synthetic activity in synthesis-deficientDNAPTaq clones.

[0197]FIG. 18A depicts the substrate molecule (SEQ ID NOS: 15 and 17)used to test the ability of synthesis-deficient DNAPs to cleave shorthairpin structures.

[0198]FIG. 18B shows an autoradiogram of a gel resolving the products ofa cleavage reaction run using the substrate shown in FIG. 19A.

[0199]FIG. 19 provides the complete 206-mer duplex sequence (SEQ ID NO:27) employed as a substrate for the 5′ nucleases of the presentinvention

[0200]FIGS. 20A and B show the cleavage of linear nucleic acidsubstrates (based on the 206-mer of FIG. 21) by wild type DNAPs and 5′nucleases isolated from Thermus aquaticus and Thermus flavus.

[0201]FIG. 21A shows the “nibbling” phenomenon detected with the DNAPsof the present invention.

[0202]FIG. 21B shows that the “nibbling” of FIG. 25A is 5′ nucleolyticcleavage and not phosphatase cleavage.

[0203]FIGS. 22A and 22B show schematic representations ofsingle-stranded and duplex substrates, and demonstrate that the“nibbling” phenomenon is duplex dependent.

[0204]FIG. 23 is a schematic showing how “nibbling” can be employed in adetection assay.

[0205]FIGS. 24A and B demonstrates that “nibbling” can be targetdirected.

[0206]FIG. 25 provides a schematic drawing of a target nucleic acid withan INVADER oligonucleotide and a probe oligonucleotide annealed to thetarget.

[0207]FIG. 26 provides a schematic showing the S-60 hairpinoligonucleotide (SEQ ID NO: 29) with the annealed P-15 oligonucleotide(SEQ ID NO: 30).

[0208]FIG. 27 is an autoradiogram of a gel showing the results of acleavage reaction run using the S-60 hairpin in the presence or absenceof the P-15 oligonucleotide.

[0209] FIGS. 28A-C provides schematic diagrams showing three differentarrangements of target-specific oligonucleotides and their hybridizationto a target nucleic acid which also has a probe oligonucleotide annealedthereto (SEQ ID NOS: 31-35).

[0210]FIG. 29 is the image generated by a fluorescence imager showingthat the presence of an INVADER oligonucleotide causes a shift in thesite of cleavage in a probe/target duplex.

[0211]FIG. 30 is the image generated by a fluorescence imager showingthe products of INVADER oligonucleotide-directed cleavage assays runusing the three target-specific oligonucleotides diagrammed in FIG. 28.

[0212]FIG. 31 is the image generated by a fluorescence imager showingthe products of INVADER oligonucleotide-directed cleavage assays run inthe presence or absence of non-target nucleic acid molecules.

[0213]FIG. 32 is the image generated by a fluorescence imager showingthe products of INVADER oligonucleotide-directed cleavage assays run inthe presence of decreasing amounts of target nucleic acid.

[0214]FIG. 33 is the image generated by a fluorescence imager showingthe products of INVADER oligonucleotide-directed cleavage assays run inthe presence or absence of saliva extract using various thermostable 5′nucleases or DNA polymerases.

[0215]FIG. 34 is the image generated by a fluorescence imager showingthe products of INVADER oligonucleotide-directed cleavage assays runusing various 5′ nucleases.

[0216]FIG. 35 is the image generated by a fluorescence imager showingthe products of INVADER oligonucleotide-directed cleavage assays runusing two target nucleic acids which differ by a single basepair at twodifferent reaction temperatures.

[0217]FIG. 36A provides a schematic showing the effect of elevatedtemperature upon the annealing and cleavage of a probe oligonucleotidealong a target nucleic acid wherein the probe contains a region ofnoncomplementarity with the target.

[0218]FIG. 36B provides a schematic showing the effect of adding anupstream oligonucleotide upon the annealing and cleavage of a probeoligonucleotide along a target nucleic acid wherein the probe contains aregion of noncomplementarity with the target.

[0219]FIG. 37 provides a schematic showing an arrangement of atarget-specific INVADER oligonucleotide (SEQ ID NO: 39) and atarget-specific probe oligonucleotide (SEQ ID NO: 38) bearing a 5′Cy3label along a target nucleic acid (SEQ ID NO: 31).

[0220]FIG. 38 is the image generated by a fluorescence imager showingthe products of INVADER oligonucleotide-directed cleavage assays run inthe presence of increasing concentrations of KCl.

[0221]FIG. 39 is the image generated by a fluorescence imager showingthe products of INVADER oligonucleotide-directed cleavage assays run inthe presence of increasing concentrations of MnCl₂ or MgCl₂.

[0222]FIG. 40 is the image generated by a fluorescence imager showingthe products of INVADER oligonucleotide-directed cleavage assays run inthe presence of increasing amounts of genomic DNA or tRNA.

[0223]FIG. 41 is the image generated by a fluorescence imager showingthe products of INVADER oligonucleotide-directed cleavage assays run usea HCV RNA target.

[0224]FIGS. 42A and 42B are images generated by a fluorescence imagershowing the products of INVADER oligonucleotide-directed cleavage assaysrun using a HCV RNA target and demonstrate the stability of RNA targetsunder INVADER oligonucleotide-directed cleavage assay conditions.

[0225]FIG. 43 is the image generated by a fluorescence imager showingthe sensitivity of detection and the stability of RNA in INVADERoligonucleotide-directed cleavage assays run using a HCV RNA target.

[0226]FIG. 44 is the image generated by a fluorescence imager showingthermal degradation of oligonucleotides containing or lacking a 3′phosphate group.

[0227]FIG. 45 depicts the structure of amino-modified oligonucleotides70 and 74.

[0228]FIG. 46 depicts the structure of amino-modified oligonucleotide 75

[0229]FIG. 47 depicts the structure of amino-modified oligonucleotide76.

[0230]FIG. 48 is the image generated by a fluorescence imager scan of anIEF gel showing the migration of substrates 70, 70 dp, 74, 74 dp, 75, 75dp, 76 and 76 dp.

[0231]FIG. 49A provides a schematic showing an arrangement of atarget-specific INVADER oligonucleotide (SEQ ID NO: 50) and atarget-specific probe oligonucleotide (SEQ ID NO: 51) bearing a 5′Cy3label along a target nucleic acid (SEQ ID NO: 52).

[0232]FIG. 49B is the image generated by a fluorescence imager showingthe detection of specific cleavage products generated in an invasivecleavage assay using charge reversal (i.e., charge based separation ofcleavage products).

[0233]FIG. 50 is the image generated by a fluorescence imager whichdepicts the sensitivity of detection of specific cleavage productsgenerated in an invasive cleavage assay using charge reversal.

[0234]FIG. 51 depicts a first embodiment of a device for thecharge-based separation of oligonucleotides.

[0235]FIG. 52 depicts a second embodiment of a device for thecharge-based separation of oligonucleotides.

[0236]FIG. 53 shows an autoradiogram of a gel showing the results ofcleavage reactions run in the presence or absence of a primeroligonucleotide; a sequencing ladder is shown as a size marker.

[0237] FIGS. 54A-D depict four pairs of oligonucleotides; in each pairshown, the upper arrangement of a probe annealed to a target nucleicacid lacks an upstream oligonucleotide and the lower arrangementcontains an upstream oligonucleotide (SEQ ID NOS: 32 and 54-58 are shownin FIGS. 54A-D).

[0238]FIG. 55 shows the chemical structure of several positively chargedheterodimeric DNA-binding dyes.

[0239]FIG. 56 is a schematic showing alternative methods for the tailingand detection of specific cleavage products in the context of theINVADER oligonucleotide-directed cleavage assay.

[0240]FIG. 57 provides a schematic drawing of a target nucleic acid withan INVADER oligonucleotide, a miniprobe, and a stacker oligonucleotideannealed to the target.

[0241]FIG. 58 provides a space-filling model of the 3-dimensionalstructure of the T5 5′-exonuclease.

[0242] FIGS. 59A-E provides an alignment of the amino acid sequences ofseveral FEN-1 nucleases including the Methanococcus jannaschii FEN-1protein (MJAFEN1.PRO), the Pyrococcus furiosus FEN-1 protein(PFUFEN1.PRO), the human FEN-1 protein (HUMFEN1.PRO), the mouse FEN-1protein (MUSFEN1.PRO), the Saccharomyces cerevisiae YKL510 protein(YST510.PRO), the Saccharomyces cerevisiae RAD2 protein (YSTRAD2.PRO),the Shizosaccharomyces pombe RAD13 protein (SPORAD13.PRO), the human XPGprotein (HUMXPG.PRO), the mouse XPG protein (MUSXPG.PRO), the Xenopuslaevis XPG protein (XENXPG.PRO) and the C. elegans RAD2 protein(CELRAD2.PRO) (SEQ ID NOS: 135-145, respectively); portions of the aminoacid sequence of some of these proteins were not shown in order tomaximize the alignment between proteins (specifically, amino acids 122to 765 of the YSTRAD2 sequence were deleted; amino acids 122 to 746 ofthe SPORAD13 sequence were deleted; amino acids 122 to 757 of the HUMXPGsequence were deleted; amino acids 122 to 770 of the MUSXPG sequencewere deleted; and amino acids 122 to 790 of the XENXPG sequence weredeleted). The numbers to the left of each line of sequence refers to theamino acid residue number; dashes represent gaps introduced to maximizealignment.

[0243]FIG. 60 is a schematic showing the S-33 (SEQ ID NO: 84) and 11-8-0(SEQ ID NO: 85) oligonucleotides in a folded configuration; the cleavagesite is indicated by the arrowhead.

[0244]FIG. 61 shows a Coomassie stained SDS-PAGE gel showing thethrombin digestion of CLEAVASE BN/thrombin.

[0245]FIG. 62 is the image generated by a fluorescence imager showingthe products produced by the cleavage of the S-60 hairpin using CLEAVASEBN/thrombin (before and after thrombin digestion).

[0246]FIG. 63 is the image generated by a fluorescence imager showingthe products produced by the cleavage of circular M13 DNA using CLEAVASEBN/thrombin.

[0247]FIG. 64 is an SDS-PAGE gel showing the migration of purifiedCLEAVASE BN nuclease, Pfu FEN-1, Pwo FEN-1 and Mja FEN-1.

[0248]FIG. 65 is the image generated by a fluorescence imager showingthe products produced by the cleavage of the S-33 and 11-8-0oligonucleotides by CLEAVASE BN and the Mja FEN-1 nucleases.

[0249]FIG. 66 is the image generated by a fluorescence imager showingthe products produced by the incubation of an oligonucleotide eitherhaving or lacking a 3′-OH group with TdT.

[0250]FIG. 67 is the image generated by a fluorescence imager showingthe products produced the incubation of cleavage products with TdT.

[0251]FIG. 68 is a photograph of a Universal GeneComb™ card showing thecapture and detection of cleavage products on a nitrocellulose support.

[0252]FIG. 69 is the image generated by a fluorescence imager showingthe products produced using the CLEAVASE A/G and Pfu FEN-1 nucleases anda fluorescein-labeled probe.

[0253]FIG. 70 is the image generated by a fluorescence imager showingthe products produced using the CLEAVASE A/G and Pfu FEN-1 nucleases anda Cy3-labeled probe.

[0254]FIG. 71 is the image generated by a fluorescence imager showingthe products produced using the CLEAVASE A/G and Pfu FEN-1 nucleases anda TET-labeled probe.

[0255]FIGS. 72A and 72B are images generated by a fluorescence imagershowing the products produced using the CLEAVASE A/G and Pfu FEN-1nucleases and probes having or lacking a 5′ positive charge; the gelshown in FIG. 83A was run in the standard direction and the gel shown inFIG. 84B was run in the reverse direction.

[0256]FIG. 73 shows the structure of 3-nitropyrrole and 5-nitroindole.

[0257]FIG. 74 shows the sequence of oligonucleotides 109, 61 and 67 (SEQID NOS: 97, 50 and 51) annealed into a cleavage structure as well as thesequence of oligonucleotide 67 (SEQ ID NO: 51) and a composite of SEQ IDNOS: 98, 99, 101 and 102.

[0258] FIGS. 75A-C show images generated by a fluorescence imagershowing the products produced in an INVADER oligonucleotide-directedcleavage assay performed at various temperatures using a miniprobe whichis either completely complementary to the target or contains a singlemismatch with the target.

[0259]FIG. 76 shows the sequence of oligonucleotides 166 (SEQ ID NO:103), 165 (SEQ ID NO: 104), 161 (SEQ ID NO: 106), 162 (SEQ ID NO: 105)and 164 (SEQ ID NO: 107) as well as a cleavage structure.

[0260]FIG. 77 shows the image generated by a fluorescence imager showingthe products produced in an INVADER oligonucleotide-directed cleavageassay performed using ras gene sequences as the target.

[0261] FIGS. 78A-C show the sequence of the S-60 hairpin (SEQ ID NO: 29)(A), and the P-15 oligonucleotide (SEQ ID NO: 30) (shown annealed to theS-60 hairpin in B) and the image generated by a fluorescence imagershowing the products produced by cleavage of the S-60 hairpin in thepresence of various INVADER oligonucleotides.

[0262]FIG. 79 shows the structure of various 3′ end substituents.

[0263]FIG. 80 is a composite graph showing the effect of probeconcentration, temperature and a stacker oligonucleotide on the cleavageof miniprobes.

[0264]FIG. 81 shows the sequence of the IT-2 oligonucleotide (SEQ ID NO:115; shown in a folded configuration) as well as the sequence of theIT-1 (SEQ ID NO: 116) and IT-A (SEQ ID NO: 117) oligonucleotides.

[0265]FIG. 82 shows the image generated by a fluorescence imager showingthe products produced by cleavage of the oligonucleotides shown in FIG.92 by CLEAVASE A/G nuclease.

[0266]FIG. 83 shows the image generated by a fluorescence imager whichprovides a comparison of the rates of cleavage by the Pfu FEN-1 and MjaFEN-1 nucleases.

[0267]FIG. 84 shows the image generated by a fluorescence imager whichdepicts the detection of RNA targets using a miniprobe and stackeroligonucleotides.

[0268] FIGS. 85A-C provide schematics showing particular embodiments ofthe present invention wherein a T7 promoter region and copy templateannealed with either no oligonucleotide (A), a complete promoteroligonucleotide (B) or a complete promoter oligonucleotide with a 3′tail (C); one strand of the T7 promoter region is indicated by thehatched line.

[0269] FIGS. 86A-D provide schematics showing particular embodiments ofthe present invention wherein a T7 promoter region and copy templateannealed with either a cut probe(A), a partial promoter oligonucleotide(B), an uncut oligonucleotide (C) or both an uncut probe and a partialpromoter oligonucleotide (D).

[0270]FIG. 87 provides a schematic illustrating one embodiment of thepresent invention wherein a template-dependent DNA polymerase is used toextend a cut probe to complete a T7 promoter region and thereby allowtranscription.

[0271]FIGS. 88A and 88B provide schematic diagrams illustrating that anuncut probe combined with a partial promoter oligonucleotide does notpermit transcription while a cut probe combined with a partial promoteroligonucleotide generates a complete (but nicked) promoter whichsupports transcription.

[0272]FIG. 89 shows the image generated by a fluorescence imager whichshows that primer extension can be used to complete a partial promoterformed by a cut probe (lanes 1-5) and that annealing a cut probegenerated in an invasive cleavage assay can complete a partial T7promoter to permit transcription (lanes 6-9).

[0273] FIGS. 90A-C provide schematics showing particular embodiments ofthe present invention which illustrate that the use of a partialpromoter oligonucleotide with a paired 5′ tail can be used to blocktranscription from a composite promoter formed by the annealing of anuncut probe.

[0274]FIG. 91 shows the image generated by a fluorescence imager whichshows that transcription from a “leaky” branched T7 composite promotercan be shut down by the use of a downstream partial promoteroligonucleotide having a paired 5′ tail.

[0275]FIG. 92 shows the image generated by a fluorescence imager whichshows that the location of the nick site in a nicked composite T7promoter can effect the efficiency of transcription.

[0276] FIGS. 93A-C are schematics showing the nucleic acids tested inreactions 1-4 of FIG. 93D; these schematics show SEQ ID NOS: 123-125.FIG. 93D shows the image generated by a fluorescence imager, which showsthat the presence of an unpaired 3′ tail on a full-length promoteroligonucleotide decreases but does not abolish transcription.

[0277]FIG. 94 is a schematic which illustrates one embodiment of thepresent invention where a composite T7 promoter region is created by thebinding of the cut probe oligonucleotide downstream of the partialpromoter oligo.

[0278] FIGS. 95A-D provide schematics showing particular embodiments ofthe present invention which show various ways in which a compositepromoter can be formed wherein the nick is located in the template (orbottom) strand.

[0279]FIG. 96 is a schematic which illustrates one embodiment of thepresent invention where the cut probe from an initial invasive cleavagereaction is employed as the INVADER oligonucleotide in a second invasivecleavage reaction.

[0280]FIG. 97 is a schematic which illustrates one embodiment of thepresent invention where the cut probe from an initial invasive cleavagereaction is employed as an integrated INVADER-target complex in a secondinvasive cleavage reaction.

[0281]FIG. 98 shows the nucleotide sequence of the PR1 probe (SEQ ID NO:119), the IT3 INVADER-Target oligonucleotide (SEQ ID NO: 118), theIT3-8, IT3-6, IT3-4, IT3-3 and IT3-0 oligonucleotides (SEQ ID NOS:147-151, respectively).

[0282] FIGS. 99A-E depict structures that may be employed to determinethe ability of an enzyme to cleave a probe in the presence and theabsence of an upstream oligonucleotide. FIG. 99 displays the sequence ofoligonucleotide 89-15-1 (SEQ ID NO: 152), oligonucleotide 81-69-5 (SEQID NO: 156), oligonucleotide 81-69-4 (SEQ ID NO 155), oligonucleotide81-69-3 (SEQ ID NO: 154), oligonucleotide 81-69-2 (SEQ ID NO: 153) and aportion of M13mp18 (SEQ ID NO: 163).

[0283]FIG. 100 shows the image generated by a fluorescence imager whichshows the dependence of Pfu FEN-1 on the presence of an overlappingupstream oligonucleotide for specific cleavage of the probe.

[0284]FIG. 101a shows the image generated by a fluorescence imager whichcompares the amount of product generated in a standard (i.e., anon-sequential invasive cleavage reaction) and a sequential invasivecleavage reaction.

[0285]FIG. 101b is a graph comparing the amount of product generated ina standard or basic (i.e., a non-sequential invasive cleavage reaction)and a sequential invasive cleavage reaction (“invader sqrd”) (yaxis=fluorescence units; x axis=attomoles of target).

[0286]FIG. 102 shows the image generated by a fluorescence imager whichshows that the products of a completed sequential invasive cleavagereaction cannot cross contaminant a subsequent similar reaction.

[0287]FIG. 103 shows the sequence of the oligonucleotide employed in aninvasive cleavage reaction for the detection of HCMV viral DNA; FIG. 103shows the sequence of oligonucleotide 89-76 (SEQ ID NO: 161),oligonucleotide 89-44 (SEQ ID NO: 160) and nucleotides 3057-3110 of theHCMV genome (SEQ ID NO: 162).

[0288]FIG. 104 shows the image generated by a fluorescence imager whichshows the sensitive detection of HCMV viral DNA in samples containinghuman genomic DNA using an invasive cleavage reaction.

[0289]FIG. 105 is a schematic which illustrates one embodiment of thepresent invention, where the cut probe from an initial invasive cleavagereaction is employed as the INVADER oligonucleotide in a second invasivecleavage reaction, and where an ARRESTOR oligonucleotide preventsparticipation of remaining uncut first probe in the cleavage of thesecond probe.

[0290]FIG. 106 is a schematic which illustrates one embodiment of thepresent invention, where the cut probe from an initial invasive cleavagereaction is employed as an integrated INVADER-target complex in a secondinvasive cleavage reaction, and where an ARRESTOR oligonucleotideprevents participation of remaining uncut first probe in the cleavage ofthe second probe.

[0291]FIG. 107 shows three images generated by a fluorescence imagershowing that two different lengths of 2′O-methyl, 3′ terminalamine-modified ARRESTOR oligonucleotide both reduce non-specificbackground cleavage of the secondary probe when included in the secondstep of a reaction where the cut probe from an initial invasive cleavagereaction is employed as an integrated INVADER-target complex in a secondinvasive cleavage reaction.

[0292]FIG. 108A shows two images generated by a fluorescence imagershowing the effects on nonspecific and specific cleavage signal ofincreasing concentrations of primary probe in the first step of areaction where the cut probe from an initial invasive cleavage reactionis employed as the INVADER oligonucleotide in a second invasive cleavagereaction.

[0293]FIG. 108B shows two images generated by a fluorescence imagershowing the effects on nonspecific and specific cleavage signal ofincreasing concentrations of primary probe in the first step of areaction, and inclusion of a 2′O-methyl, 3′ terminal amine-modifiedARRESTOR oligonucleotide in the second step of a reaction where the cutprobe from an initial invasive cleavage reaction is employed as theINVADER oligonucleotide in a second invasive cleavage reaction.

[0294]FIG. 108C shows shows a graph generated using the spreadsheetMicrosoft Excel software, comparing the effects on nonspecific andspecific cleavage signal of increasing concentrations of primary probein the first step of a reaction, in the presence or absence of a2′O-methyl, 3′ terminal amine-modified ARRESTOR oligonucleotide in thesecond step of a reaction where the cut probe from an initial invasivecleavage reaction is employed as the INVADER oligonucleotide in a secondinvasive cleavage reaction.

[0295]FIG. 109A shows two images generated by a fluorescence imagershowing the effects on nonspecific and specific cleavage signal ofincluding an unmodified ARRESTOR oligonucleotide in the second step of areaction where the cut probe from an initial invasive cleavage reactionis employed as the INVADER oligonucleotide in a second invasive cleavagereaction.

[0296]FIG. 109B shows two images generated by a fluorescence imagershowing the effects on nonspecific and specific cleavage signal ofincluding a 3′ terminal amine modified ARRESTOR, a partially 2′O-methylsubstituted, 3′ terminal amine modified ARRESTOR oligonucleotide, or anentirely 2′O-methyl, 3′ terminal amine modified ARRESTOR oligonucleotidein the second step of a reaction where the cut probe from an initialinvasive cleavage reaction is employed as the INVADER oligonucleotide ina second invasive cleavage reaction.

[0297]FIG. 110A shows two images generated by a fluorescence imagercomparing the effects on nonspecific and specific cleavage signal ofincluding an ARRESTOR oligonucleotides of different lengths in thesecond step of a reaction where the cut probe from an initial invasivecleavage reaction is employed as the INVADER oligonucleotide in a secondinvasive cleavage reaction.

[0298]FIG. 110B shows two images generated by a fluorescence imagercomparing the effects on nonspecific and specific cleavage signal ofincluding an ARRESTOR oligonucleotides of different lengths in thesecond step of a reaction where the cut probe from an initial invasivecleavage reaction is employed as the INVADER oligonucleotide in a secondinvasive cleavage reaction, and in which a longer variant of thesecondary probe used in the reactions in FIG. 110A is tested.

[0299]FIG. 110C shows a schematic diagram of a primary probe alignedwith several ARRESTOR oligonucleotides of different lengths. The regionof the primary probe that is complementary to the HBV target sequence isunderlined. The ARRESTOR oligonucleotides are aligned with the probe bycomplementarity.

[0300]FIG. 111 shows two images generated by a fluorescence imagercomparing the effects on nonspecific and specific cleavage signal ofincluding ARRESTOR oligonucleotides of different lengths in the secondstep of a reaction where the cut probe from an initial invasive cleavagereaction is employed as the INVADER oligonucleotide in a second invasivecleavage reaction, using secondary probes of two different lengths.

[0301]FIG. 112A provides a schematic diagram that illustrates oneembodiment of the present invention wherein the cut probe from aninitial invasive cleavage reaction is employed as the INVADERoligonucleotide in a second invasive cleavage reaction using a FRETcassette. The region indicated as “N” is the overlap required forcleavage in this embodiment. 112B diagrams how a mismatch between theprobe and the target strand at position “N” disrupts the overlap,thereby suppressing cleavage of the probe.

[0302]FIG. 113A shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 195), probe oligonucleotide (SEQ ID NO: 197) and FRETcassette (SEQ ID NO: 201) for the detection of the Apo E 112 arg allele.

[0303]FIG. 113B shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 195), probe oligonucleotide (SEQ ID NO: 198) and FRETcassette for the detection (SEQ ID NO: 201) of the Apo E 112 cys allele.

[0304]FIG. 113C shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 196), probe oligonucleotide (SEQ ID NO: 199) and FRETcassette (SEQ ID NO: 201) for the detection of the Apo E 158 arg allele.

[0305]FIG. 113D shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 196), probe oligonucleotide (SEQ ID NO: 200) and FRETcassette (SEQ ID NO: 201) for the detection of the Apo E 158 cys allele.

[0306]FIG. 114A provides a bar graph showing the detection of the argand cys alleles at the Apo E 112 locus in 2 synthetic controls and 5samples of human genomic DNA.

[0307]FIG. 114B provides a bar graph showing the detection of the argand cys alleles at the Apo E 158 locus in 2 synthetic controls and 5samples of human genomic DNA.

[0308]FIG. 115A shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 202), probe oligonucleotide (SEQ ID NO: 208) and FRETcassette (SEQ ID NO: 210) for the detection of the wild-type C282 alleleof the human HFE gene.

[0309]FIG. 115B shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 202), probe oligonucleotide (SEQ ID NO: 209) and FRETcassette (SEQ ID NO: 210) for the detection of the C282Y mutant alleleof the human HFE gene.

[0310]FIG. 115C shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 203), probe oligonucleotide (SEQ ID NO: 211) and FRETcassette (SEQ ID NO: 206) for the detection of the wild-type H63 alleleof the human HFE gene.

[0311]FIG. 115D shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 203), probe oligonucleotide (SEQ ID NO: 212) and FRETcassette (SEQ ID NO: 213) for the detection of the H63D mutant allele ofthe human HFE gene.

[0312]FIG. 116 provides a bar graph showing the analysis of the C282Y(first set of eight tests, left to right) and H63D (second set of eighttests, left to right) mutations in the human HFE gene, each tested in 2synthetic controls and 5 samples of human genomic DNA.

[0313]FIG. 117A shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 216), probe oligonucleotide (SEQ ID NO: 217) and FRETcassette (SEQ ID NO: 225) for the detection of the wild-type allele atposition 677 of the human MTHFR gene.

[0314]FIG. 117B shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 216), probe oligonucleotide (SEQ ID NO: 218) and FRETcassette (SEQ ID NO: 225) for the detection of the mutant allele atposition 677 of the human MTHFR gene.

[0315]FIG. 118 provides a bar graph showing the analysis of the C677Tmutation in the human MTHFR gene in 3 synthetic control samples and 3samples of human genomic DNA.

[0316]FIG. 119A shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 222), probe oligonucleotide (SEQ ID NO: 223) and FRETcassette (SEQ ID NO: 225) for the detection of the wild-type allele atposition 20210 of the human prothrombin gene.

[0317]FIG. 119B shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 222), probe oligonucleotide (SEQ ID NO: 224) and FRETcassette (SEQ ID NO: 225) for the detection of the mutant allele atposition 20210 of the human prothrombin gene.

[0318]FIG. 120 provides a bar graph showing the analysis of the A2020Gmutation in the human prothrombin gene in 2 synthetic control samplesand 3 samples of human genomic DNA.

[0319]FIG. 121A shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 228), probe oligonucleotide (SEQ ID NO: 229) and FRETcassette (SEQ ID NO: 230) for the detection of the R-2 mutant allele ofthe human factor V gene.

[0320]FIG. 121B shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 231), probe oligonucleotide (SEQ ID NO: 232) and FRETcassette (SEQ ID NO: 230) for the detection of the human α-actin gene.

[0321]FIG. 122 provides a bar graph showing the detection of the R-2mutant (HR-2) of the human factor V gene, compared to the detection ofthe internal control (IC), the α-actin gene, 3 synthetic control samplesand 2 samples of human genomic DNA.

[0322]FIG. 123A shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 235), probe oligonucleotide (SEQ ID NO: 236) and FRETcassette (SEQ ID NO: 225) for the detection of the wild-type allele atposition −308 in the promoter of the human TNF-α gene.

[0323]FIG. 123B shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 235), probe oligonucleotide (SEQ ID NO: 237) and FRETcassette (SEQ ID NO: 225) for the detection of the mutant allele atposition −308 in the promoter of the human TNF-α gene.

[0324]FIG. 124 provides a bar graph showing the analysis of the −308mutation in the promoter of the human TNF-α gene in 3 synthetic controlsamples and 3 samples of human genomic DNA.

[0325]FIG. 125A shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 240), probe oligonucleotide (SEQ ID NO: 241) and FRETcassette (SEQ ID NO: 225) for the detection of the wild-type allele atcodon position 506 of the human factor V gene.

[0326]FIG. 125B shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 240), probe oligonucleotide (SEQ ID NO: 242) and FRETcassette (SEQ ID NO: 225) for the detection of the A506G mutant alleleof the human factor V gene.

[0327]FIG. 126 provides a bar graph showing the analysis of the A506Gmutation in the human factor V gene in 3 synthetic control samples and 6samples of human genomic DNA.

[0328]FIG. 127A shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 243), probe oligonucleotide (SEQ ID NO: 244) and FRETcassette (SEQ ID NO: 245) for the detection of the mecA gene associatedwith methicillin resistance in S. aureus.

[0329]FIG. 127B shows a schematic diagram of an INVADER oligonucleotide(SEQ ID NO: 246), probe oligonucleotide (SEQ ID NO: 247) and FRETcassette (SEQ ID NO: 245) for the detection of the nuc gene, aspecies-specific gene that distinguishes S. aureus from S. haemolyticus.

[0330]FIG. 128 provides a bar graph showing the detection of the mecAgene, compared to the detection of the S. aureus-specific nuc gene inDNA from methicillin-sensitive S. aureus (MSSA), methicillin-resistantS. aureus (MRSA), S. haemolyticus, and amplified control targets for themecA and nuc target sequences.

DESCRIPTION OF THE INVENTION

[0331] Introduction:

[0332] The present invention relates to methods and compositions fortreating nucleic acid, and in particular, methods and compositions fordetection and characterization of nucleic acid sequences and sequencechanges.

[0333] In preferred embodiments, the present invention relates to meansfor cleaving a nucleic acid cleavage structure in a site-specificmanner. While the present invention provides a variety of cleavageagents, in some embodiments, the present invention relates to a cleavingenzyme having 5′ nuclease activity without interfering nucleic acidsynthetic ability. In other embodiments, the present invention providesnovel polymerases (e.g., thermostable polymerases) possessing alteredpolymerase and/or nucleases activities.

[0334] For example, in some embodiments, the present invention provides5′ nucleases derived from thermostable DNA polymerases that exhibitaltered DNA synthetic activity from that of native thermostable DNApolymerases. The 5′ nuclease activity of the polymerase is retainedwhile the synthetic activity is reduced or absent. Such 5′ nucleases arecapable of catalyzing the structure-specific cleavage of nucleic acidsin the absence of interfering synthetic activity. The lack of syntheticactivity during a cleavage reaction results in nucleic acid cleavageproducts of uniform size.

[0335] The novel properties of the nucleases of the invention form thebasis of a method of detecting specific nucleic acid sequences. Thismethod relies upon the amplification of the detection molecule ratherthan upon the amplification of the target sequence itself as do existingmethods of detecting specific target sequences.

[0336] DNA polymerases (DNAPs), such as those isolated from E. coli orfrom thermophilic bacteria of the genus Thermus as well as otherorganisms, are enzymes that synthesize new DNA strands. Several of theknown DNAPs contain associated nuclease activities in addition to thesynthetic activity of the enzyme.

[0337] Some DNAPs are known to remove nucleotides from the 5′ and 3′ends of DNA chains (Komberg, DNA Replication, W. H. Freeman and Co., SanFrancisco, pp. 127-139 [1980]). These nuclease activities are usuallyreferred to as 5′ exonuclease and 3′ exonuclease activities,respectively. For example, the 5′ exonuclease activity located in theN-terminal domain of several DNAPs participates in the removal of RNAprimers during lagging strand synthesis during DNA replication and theremoval of damaged nucleotides during repair. Some DNAPs, such as the E.coli DNA polymerase (DNAPEc1), also have a 3′ exonuclease activityresponsible for proof-reading during DNA synthesis (Komberg, supra).

[0338] A DNAP isolated from Thermus aquaticus, termed Taq DNA polymerase(DNAPTaq), has a 5′ exonuclease activity, but lacks a functional 3′exonucleolytic domain (Tindall and Kunkell, Biochem., 27:6008 [1988]).Derivatives of DNAPEc1 and DNAPTaq, respectively called the Klenow andStoffel fragments, lack 5′ exonuclease domains as a result of enzymaticor genetic manipulations (Brutlag et al., Biochem. Biophys. Res.Commun., 37:982 [1969]; Erlich et al., Science 252:1643 [1991]; Setlowand Kornberg, J. Biol. Chem., 247:232 [1972]).

[0339] The 5′ exonuclease activity of DNAPTaq was reported to requireconcurrent synthesis (Gelfand, PCR Technology—Principles andApplications for DNA Amplification, H. A. Erlich, [Ed.], Stockton Press,New York, p. 19 [1989]). Although mononucleotides predominate among thedigestion products of the 5′ exonucleases of DNAPTaq and DNAPEc1, shortoligonucleotides (≦12 nucleotides) can also be observed implying thatthese so-called 5′ exonucleases can function endonucleolytically(Setlow, supra; Holland et al., Proc. Natl. Acad. Sci. USA 88:7276[1991]).

[0340] In WO 92/06200, Gelfand et al. show that the preferred substrateof the 5′ exonuclease activity of the thermostable DNA polymerases isdisplaced single-stranded DNA. Hydrolysis of the phosphodiester bondoccurs between the displaced single-stranded DNA and the double-helicalDNA with the preferred exonuclease cleavage site being a phosphodiesterbond in the double helical region. Thus, the 5′ exonuclease activityusually associated with DNAPs is a structure-dependent single-strandedendonuclease and is more properly referred to as a 5′ nuclease.Exonucleases are enzymes that cleave nucleotide molecules from the endsof the nucleic acid molecule. Endonucleases, on the other hand, areenzymes that cleave the nucleic acid molecule at internal rather thanterminal sites. The nuclease activity associated with some thermostableDNA polymerases cleaves endonucleolytically but this cleavage requirescontact with the 5′ end of the molecule being cleaved. Therefore, thesenucleases are referred to as 5′ nucleases.

[0341] When a 5′ nuclease activity is associated with a eubacterial TypeA DNA polymerase, it is found in the one third N-terminal region of theprotein as an independent functional domain. The C-terminal two-thirdsof the molecule constitute the polymerization domain that is responsiblefor the synthesis of DNA. Some Type A DNA polymerases also have a 3′exonuclease activity associated with the two-third C-terminal region ofthe molecule.

[0342] The 5′ exonuclease activity and the polymerization activity ofDNAPs can be separated by proteolytic cleavage or genetic manipulationof the polymerase molecule. The Klenow or large proteolytic cleavagefragment of DNAPEc1 contains the polymerase and 3′ exonuclease activitybut lacks the 5′ nuclease activity. The Stoffel fragment of DNAPTaq(DNAPStf) lacks the 5′ nuclease activity due to a genetic manipulationthat deleted the N-terminal 289 amino acids of the polymerase molecule(Erlich et al., Science 252:1643 [1991]). WO 92/06200 describes athermostable DNAP with an altered level of 5′ to 3′ exonuclease. U.S.Pat. No. 5,108,892 describes a Thermus aquaticus DNAP without a 5′ to 3′exonuclease. Thermostable DNA polymerases with lessened amounts ofsynthetic activity are available (Third Wave Technologies, Madison,Wis.) and are described in U.S. Pat. Nos. 5,541,311, 5,614,402,5,795,763, 5,691,142, and 5,837,450, herein incorporated by reference intheir entireties. The present invention provides 5′ nucleases derivedfrom thermostable Type A DNA polymerases that retain 5′ nucleaseactivity but have reduced or absent synthetic activity. The ability touncouple the synthetic activity of the enzyme from the 5′ nucleaseactivity proves that the 5′ nuclease activity does not requireconcurrent DNA synthesis as was previously reported (Gelfand, PCRTechnology, supra).

[0343] In addition to the 5′-exonuclease domains of the DNA polymerase Iproteins of Eubacteria, described above, 5′ nucleases have been foundassociated with bacteriophage, eukaryotes and archaebacteria. Overall,all of the enzymes in this family display very similar substratespecificities, despite their limited level of sequence similarity.Consequently, enzymes suitable for use in the methods of the presentinvention may be isolated or derived from a wide array of sources.

[0344] A mammalian enzyme with functional similarity to the5′-exonuclease domain of E. coli Pol I was isolated nearly 30 years ago(Lindahl, et al., Proc Natl Acad Sci U S A 62(2): 597-603 [1969]).Later, additional members of this group of enzymes called flapendonucleases (FEN1) from Eukarya and Archaea were shown to possess anearly identical structure specific activity (Harrington and Lieber.Embo J 13(5), 1235-46 [1994]; Murante et al., J Biol Chem 269(2), 1191-6[1994]; Robins, et al., J Biol Chem 269(46), 28535-8 [1994]; Hosfield,et al., J Biol Chem 273(42), 27154-61 [1998), despite limited sequencesimilarity. The substrate specificities of the FEN1 enzymes, and theeubacterial and related bacteriophage enzymes have been examined andfound to be similar for all enzymes (Lyamichev, et al., Science260(5109), 778-83 [1993], Harrington and Lieber, supra, Murante, et al.,supra, Hosfield, et al, supra, Rao, et al., J Bacteriol 180(20), 5406-12[1998], Bhagwat, et al,. J. Biol Chem 272(45), 28523-30 [1997], Garforthand Sayers, Nucleic Acids Res 25(19), 3801-7 [1997]).

[0345] Using preformed substrates, many of the studies cited abovedetermined that these nucleases leave a gap upon cleavage, leading theauthors to speculate that DNA polymerase must then act to fill in thatgap to generate a ligatable nick. A number of other 5′ nucleases havebeen shown to leave a gap or overlap after cleavage of the same orsimilar flap substrates. It has since been determined that that all thestructure-specific 5′-exonucleases leave a nick after cleavage if thesubstrate has an overlap between the upstream and downstream duplexes(Kaiser et al., J. Biol .Chem. 274(30):21387-21394 [1999]). Whileduplexes having several bases of overlapping sequence can assume severaldifferent conformations through branch migration, it was determined thatcleavage occurs in the conformation where the last nucleotide at the 3′end of the upstream strand is unpaired, with the cleavage rate beingessentially the same whether the end of the upstream primer is A, C, G,or T. It was determined to be positional overlap between the 3′ end ofthe upstream primer and downstream duplex, rather then sequence overlap,that is required for optimal cleavage. In addition to allowing theseenzymes to leave a nick after cleavage, the single base of overlapcauses the enzymes to cleave several orders of magnitude faster thanwhen a substrate lacks overlap (Kaiser et al., supra).

[0346] Any of the 5′ nucleases described above may find application inone or more embodiments of the methods described herein. FEN1 nucleasesof particular utility in the methods of present invention include butare not limited to those of Methanococcus jannaschii andMethanobacterium thermoautotrophicum; particularly preferred FEN1enzymes are from Archaeoglobus fulgidus, Pyrococcus furiosus andArchaeoglobus veneficus.

[0347] The detailed description of the invention is presented in thefollowing sections:

[0348] I. Detection of Specific Nucleic Acid Sequences Using 5′Nucleases in an INVADER Directed Cleavage Assay;

[0349] II. Effect of ARRESTOR Oligonucleotides on Signal and Backgroundin Sequential Invasive Cleavage Reactions.

[0350] III. Signal Enhancement By Incorporating The Products Of AnInvasive Cleavage Reaction Into A Subsequent Invasive Cleavage Reaction;

[0351] IV. Fractionation Of Specific Nucleic Acids By Selective ChargeReversal;

[0352] V. Signal Enhancement By Tailing Of Reaction Products In TheINVADER oligonucleotide-directed Cleavage Assay;

[0353] VI. Signal Enhancement By Completion Of An Activated ProteinBinding Site;

[0354] VII. Generation of 5′ Nucleases Derived From Thermostable DNAPolymerases;

[0355] VIII. Improved Enzymes For Use In INVADERoligonucleotide-directed Cleavage Reactions;

[0356] IX. The INVADER assay for direct detection and measurement ofspecific analytes.

[0357] I. Detection of Specific Nucleic Acid Sequences Using 5′Nucleases in an INVADER Directed Cleavage Assay

[0358] 1. INVADER Assay Reaction Design

[0359] The present invention provides means for forming a nucleic acidcleavage structure that is dependent upon the presence of a targetnucleic acid and cleaving the nucleic acid cleavage structure so as torelease distinctive cleavage products. 5′ nuclease activity, forexample, is used to cleave the target-dependent cleavage structure andthe resulting cleavage products are indicative of the presence ofspecific target nucleic acid sequences in the sample. When two strandsof nucleic acid, or oligonucleotides, both hybridize to a target nucleicacid strand such that they form an overlapping invasive cleavagestructure, as described below, invasive cleavage can occur. Through theinteraction of a cleavage agent (e.g., a 5′ nuclease) and the upstreamoligonucleotide, the cleavage agent can be made to cleave the downstreamoligonucleotide at an internal site in such a way that a distinctivefragment is produced. Such embodiments have been termed the INVADERassay (Third Wave Technologies) and are described in U.S. Pat. Nos.5,846,717, 5,985,557, 5,994,069, 6,001,567, and 6,090,543, U.S. patentapplication Ser. No. 09/713,601, and PCT Publications WO 97/27214 and WO98/42873, herein incorporated by reference in their entireties.

[0360] The present invention further provides assays in which the targetnucleic acid is reused or recycled during multiple rounds ofhybridization with oligonucleotide probes and cleavage of the probeswithout the need to use temperature cycling (i.e., for periodicdenaturation of target nucleic acid strands) or nucleic acid synthesis(i.e., for the polymerization-based displacement of target or probenucleic acid strands). When a cleavage reaction is run under conditionsin which the probes are continuously replaced on the target strand (e.gthrough probe-probe displacement or through an equilibrium betweenprobe/target association and disassociation, or through a combinationcomprising these mechanisms, [The kinetics of oligonucleotidereplacement. Luis P. Reynaldo, Alexander V. Vologodskii, Bruce P. Neriand Victor I. Lyamichev. J. Mol. Biol. 97: 511-520 (2000)], multipleprobes can hybridize to the same target, allowing multiple cleavages,and the generation of multiple cleavage products.

[0361] By the extent of its complementarity to a target nucleic acidstrand, an oligonucleotide may be said to define a specific region ofsaid target. In an invasive cleavage structure, the two oligonucleotidesdefine and hybridize to regions of the target that are adjacent to oneanother (i.e., regions without any additional region of the targetbetween them). Either or both oligonucleotides may comprise additionalportions that are not complementary to the target strand. In addition tohybridizing adjacently, in order to form an invasive cleavage structure,the 3′ end of the upstream oligonucleotide must comprise an additionalmoiety. When both oligonucleotides are hybridized to a target strand toform a structure and such a 3′ moiety is present on the upstreamoligonucleotide within the structure, the oligonucleotides may be saidto overlap, and the structure may be described as an overlapping, orinvasive cleavage structure.

[0362] In one embodiment, the 3′ moiety of the invasive cleavagestructure is a single nucleotide. In this embodiment the 3′ moiety maybe any nucleotide (i.e., it may be, but it need not be complementary tothe target strand). In a preferred embodiment the 3′ moiety is a singlenucleotide that is not complementary to the target strand. In anotherembodiment, the 3′ moiety is a nucleotide-like compound (i.e., a moietyhaving chemical features similar to a nucleotide, such as a nucleotideanalog or an organic ring compound; See e.g., U.S. Pat. No. 5,985,557).In yet another embodiment the 3′ moiety is one or more nucleotides thatduplicate in sequence one or more nucleotides present at the 5′ end ofthe hybridized region of the downstream oligonucleotide. In a furtherembodiment, the duplicated sequence of nucleotides of the 3′ moiety isfollowed by a single nucleotide that is not further duplicative of thedownstream oligonucleotide sequence, and that may be any othernucleotide. In yet another embodiment, the duplicated sequence ofnucleotides of the 3′ moiety is followed by a nucleotide-like compound,as described above.

[0363] The downstream oligonucleotide may have, but need not have,additional moieties attached to either end of the region that hybridizesto the target nucleic acid strand. In a preferred embodiment, thedownstream oligonucleotide comprises a moiety at its 5′ end (i.e., a 5′moiety). In a particularly preferred embodiment, said 5′ moiety is a 5′flap or arm comprising a sequence of nucleotides that is notcomplementary to the target nucleic acid strand.

[0364] When an overlapping cleavage structure is formed, it can berecognized and cleaved by a nuclease that is specific for this structure(i.e., a nuclease that will cleave one or more of the nucleic acids inthe overlapping structure based on recognition of this structure, ratherthan on recognition of a nucleotide sequence of any of the nucleic acidsforming the structure). Such a nuclease may be termed a“structure-specific nuclease”. In some embodiments, thestructure-specific nuclease is a 5′ nuclease. In a preferred embodiment,the structure-specific nuclease is the 5′ nuclease of a DNA polymerase.In another preferred embodiment, the DNA polymerase having the 5′nuclease is synthesis-deficient. In another preferred embodiment, the 5′nuclease is a FEN-1 endonuclease. In a particularly preferredembodiment, the 5′ nuclease is thermostable.

[0365] In some embodiments, said structure-specific nucleasepreferentially cleaves the downstream oligonucleotide. In a preferredembodiment, the downstream oligonucleotide is cleaved one nucleotideinto the 5′ end of the region that is hybridized to the target withinthe overlapping structure. Cleavage of the overlapping structure at anylocation by a structure-specific nuclease produces one or more releasedportions or fragments of nucleic acid, termed “cleavage products”.

[0366] In some embodiments, cleavage of an overlapping structure isperformed under conditions wherein one or more of the nucleic acids inthe structure can disassociate (i.e. un-hybridize, or melt) from thestructure. In one embodiment, full or partial disassociation of a firstcleavage structure allows the target nucleic acid to participate in theformation of one or more additional overlapping cleavage structures. Ina preferred embodiment, the first cleavage structure is partiallydisassociated. In a particularly preferred embodiment only theoligonucleotide that is cleaved disassociates from the first cleavagestructure, such that it may be replaced by another copy of the sameoligonucleotide. In some embodiments, said disassociation is induced byan increase in temperature, such that one or more oligonucleotides canno longer hybridize to the target strand. In other embodiments, saiddisassociation occurs because cleavage of an oligonucleotide producesonly cleavage products that cannot bind to the target strand under theconditions of the reaction. In a preferred embodiment, conditions areselected wherein an oligonucleotide may associate with (i.e., hybridizeto) and disassociate from a target strand regardless of cleavage, andwherein the oligonucleotide may be cleaved when it is hybridized to thetarget as part of an overlapping cleavage structure. In a particularlypreferred embodiment, conditions are selected such that the number ofcopies of the oligonucleotide that can be cleaved when part of anoverlapping structure exceeds the number of copies of the target nucleicacid strand by a sufficient amount that when the first cleavagestructure disassociates, the probability that the target strand willassociate with an intact copy of the oligonucleotide is greater than theprobability that that it will associate with a cleaved copy of theoligonucleotide.

[0367] In some embodiments, cleavage is performed by astructure-specific nuclease that can recognize and cleave structuresthat do not have an overlap. In a preferred embodiment, cleavage isperformed by a structure-specific nuclease having a lower rate ofcleavage of nucleic acid structures that do not comprise an overlap,compared to the rate of cleavage of structures comprising an overlap. Ina particularly preferred embodiment, cleavage is performed by astructure-specific nuclease having less than 1% of the rate of cleavageof nucleic acid structures that do not comprise an overlap, compared tothe rate of cleavage of structures comprising an overlap.

[0368] In some embodiments it is desirable to detect the cleavage of theoverlapping cleavage structure. Detection may be by analysis of cleavageproducts or by analysis of one or more of the remaining uncleavednucleic acids. For convenience, the following discussion will refer tothe analysis of cleavage products, but it will be appreciated by thoseskilled in the art that these methods may as easily be applied toanalysis of the uncleaved nucleic acids in an invasive cleavagereaction. Any method known in the art for analysis of nucleic acids,nucleic acid fragments or oligonucleotides may be applied to thedetection of cleavage products.

[0369] In one embodiment, the cleavage products may be identified bychemical content, e.g., the relative amounts of each atom, eachparticular type of reactive group or each nucleotide base (Chargaff etal., J. Biol. Chem. 177: 405 [1949]) they contain. In this way, acleavage product may be distinguished from a longer nucleic acid fromwhich it was released by cleavage, or from other nucleic acids.

[0370] In another embodiment, the cleavage products may be distinguishedby a particular physical attribute, including but not limited to length,mass, charge, or charge-to-mass ratio. In yet another embodiment, thecleavage product may be distinguished by a behavior that is related to aphysical attribute, including but not limited to rate of rotation insolution, rate of migration during electrophoresis, coefficient ofsedimentation in centrifugation, time of flight in MALDI-TOF massspectrometry, migration rate or other behavior in chromatography,melting temperature from a complementary nucleic acid, orprecipitability from solution.

[0371] Detection of the cleavage products may be through release of alabel. Such labels may include, but are not limited to one or more ofany of dyes, radiolabels such as ³²P or ³⁵S, binding moieties such asbiotin, mass tags, such as metal ions or chemical groups, charge tags,such as polyamines or charged dyes, haptens such as digoxgenin,luminogenic, phosphorescent or fluorogenic moieties, and fluorescentdyes, either alone or in combination with moieties that can suppress orshift emission spectra, such as by fluorescence resonance energytransfer (FRET) or collisional fluorescence energy transfer.

[0372] In some embodiments, analysis of cleavage products may includephysical resolution or separation, for example by electrophoresis,hybridization or by selective binding to a support, or by massspectrometry methods such as MALDI-TOF. In other embodiments, theanalysis may be performed without any physical resolution or separation,such as by detection of cleavage-induced changes in fluorescence as inFRET-based analysis, or by cleavage-induced changes in the rotation rateof a nucleic acid in solution as in fluorescence polarization analysis.

[0373] Cleavage products can be used subsequently in any reaction orread-out method that can make use of oligonucleotides. Such reactionsinclude, but are not limited to, modification reactions, such asligation, tailing with a template-independent nucleic acid polymeraseand primer extension with a template-dependent nucleic acid polymerase.The modification of the cleavage products may be for purposes including,but not limited to, addition of one or more labels or binding moieties,alteration of mass, addition of specific sequences, or for any otherpurpose that would facilitate analysis of either the cleavage productsor analysis of any other by-product, result or consequence of thecleavage reaction.

[0374] Analysis of the cleavage products may involve subsequent steps orreactions that do not modify the cleavage products themselves. Forexample, cleavage products may be used to complete a functionalstructure, such as a competent promoter for in vitro transcription oranother protein binding site. Analysis may include the step of using thecompleted structure for or to perform its function. One or more cleavageproducts may also be used to complete an overlapping cleavage structure,thereby enabling a subsequent cleavage reaction, the products of whichmay be detected or used by any of the methods described herein,including the participation in further cleavage reactions.

[0375] Certain preferred embodiments of the invasive cleavage reactionsare provided in the following descriptions. As exemplified by thediagram in FIG. 29, the methods of the present invention employ at leasta pair of oligonucleotides that interact with a target nucleic acid toform a cleavage structure for a structure-specific nuclease. In someembodiments, the cleavage structure comprises i) a target nucleic acidthat may be either single-stranded or double-stranded (when adouble-stranded target nucleic acid is employed, it may be renderedsingle stranded, e.g., by heating); ii) a first oligonucleotide, termedthe “probe,” that defines a first region of the target nucleic acidsequence by being the complement of that region (regions X and Z of thetarget as shown in FIG. 29); iii) a second oligonucleotide, termed the“INVADER,” the 5′ part of which defines a second region of the sametarget nucleic acid sequence (regions Y and X in FIG. 29), adjacent toand downstream of the first target region (regions X and Z), and thesecond part of which overlaps into the region defined by the firstoligonucleotide (region X depicts the region of overlap). The resultingstructure is diagrammed in FIG. 29.

[0376] While not limiting the invention or the instant discussion to anyparticular mechanism of action, the diagram in FIG. 29 represents theeffect on the site of cleavage caused by this type of arrangement of apair of oligonucleotides. The design of such a pair of oligonucleotidesis described below in detail. In FIG. 29, the 3′ ends of the nucleicacids (i.e., the target and the oligonucleotides) are indicated by theuse of the arrowheads on the ends of the lines depicting the strands ofthe nucleic acids (and where space permits, these ends are also labeled“3′”). It is readily appreciated that the two oligonucleotides (theINVADER and the probe) are arranged in a parallel orientation relativeto one another, while the target nucleic acid strand is arranged in ananti-parallel orientation relative to the two oligonucleotides. Further,it is clear that the INVADER oligonucleotide is located upstream of theprobe oligonucleotide and that with respect to the target nucleic acidstrand, region Z is upstream of region X and region X is upstream ofregion Y (that is, region Y is downstream of region X and region X isdownstream of region Z). Regions of complementarity between the opposingstrands are indicated by the short vertical lines. While not intended toindicate the precise location of the site(s) of cleavage, the area towhich the site of cleavage within the probe oligonucleotide is shiftedby the presence of the INVADER oligonucleotide in this embodiment isindicated by the solid vertical arrowhead. An alternative representationof the target/INVADER/probe cleavage structure is shown in FIG. 32c.Neither diagram (i.e., FIG. 29 or FIG. 32c) is intended to represent theactual mechanism of action or physical arrangement of the cleavagestructure and further it is not intended that the method of the presentinvention be limited to any particular mechanism of action.

[0377] It can be considered that the binding of these oligonucleotidesin this embodiment divides the target nucleic acid into three distinctregions: one region that has complementarity to only the probe (shown as“Z”); one region that has complementarity only to the INVADERoligonucleotide (shown as “Y”); and one region that has complementarityto both oligonucleotides (shown as “X”). As discussed above, in somepreferred embodiments of the present invention, the overlap may comprisemoieties other than overlapping complementary bases. Thus, in someembodiments, the region shown as “X” can represent a region where thereis a physical, but not sequence, overlap between the INVADER and probeoligonucleotides, i.e., in these latter embodiments, there is not aregion of the target nucleic acid between regions “Z” and “Y” that hascomplementarity to both oligonucleotides.

[0378] a) Oligonucleotide Design

[0379] Design of these oligonucleotides (i.e., the INVADERoligonucleotide and the probe) is accomplished using practices that arestandard in the art. For example, sequences that have selfcomplementarity, such that the resulting oligonucleotides would eitherfold upon themselves, or hybridize to each other at the expense ofbinding to the target nucleic acid, are generally avoided.

[0380] One consideration in choosing a length for these oligonucleotidesis the complexity of the sample containing the target nucleic acid. Forexample, the human genome is approximately 3×10⁹ basepairs in length.Any 10-nucleotide sequence will appear with a frequency of 1:4¹⁰, or1:1048,576 in a random string of nucleotides, which would beapproximately 2,861 times in 3 billion basepairs. Clearly, anoligonucleotide of this length would have a poor chance of bindinguniquely to a 10 nucleotide region within a target having a sequence thesize of the human genome. If the target sequence were within a 3 kbplasmid, however, such an oligonucleotide might have a very reasonablechance of binding uniquely. By this same calculation it can be seen thatan oligonucleotide of 16 nucleotides (i.e., a 16-mer) is the minimumlength of a sequence that is mathematically likely to appear once in3×10⁹ basepairs. This level of specificity may also be provided by twoor more shorter oligonucleotides if they are configured to bind in acooperative fashion (i.e., such that they can produce the intendedcomplex only if both or all are bound to their intended targetsequences), wherein the combination of the short oligonucleotidesprovides the desired specificity. In one such embodiment, thecooperativity between the shorter oligonucleotides is by a coaxialstacking effect that can occur when the oligonucleotides hybridize toadjacent sites on a target nucleic acid. In another embodiment, theshorter oligonucleotides are connected to one another, either directly,or by one or more spacer regions. The short oligonucleotides thusconnected may bind to distal regions of the target and may be used tobridge across regions of secondary structure in a target. Examples ofsuch bridging oligonucleotides are described in PCT Publication WO98/50403, herein incorporated by reference in its entirety.

[0381] A second consideration in choosing oligonucleotide length is thetemperature range in which the oligonucleotides will be expected tofunction. A 16-mer of average base content (50% G-C bases) will have acalculated T_(m) of about 41° C., depending on, among other things, theconcentration of the oligonucleotide and its target, the salt content ofthe reaction and the precise order of the nucleotides. As a practicalmatter, longer oligonucleotides are usually chosen to enhance thespecificity of hybridization. Oligonucleotides 20 to 25 nucleotides inlength are often used, as they are highly likely to be specific if usedin reactions conducted at temperatures which are near their T_(m)s(within about 5° C. of the T_(m)). In addition, with calculated T_(m)sin the range of 50 to 70° C., such oligonucleotides (i.e., 20 to25-mers) are appropriately used in reactions catalyzed by thermostableenzymes, which often display optimal activity near this temperaturerange.

[0382] The maximum length of the oligonucleotide chosen is also based onthe desired specificity. One must avoid choosing sequences that are solong that they are either at a high risk of binding stably to partialcomplements, or that they cannot easily be dislodged when desired (e.g.,failure to disassociate from the target once cleavage has occurred orfailure to disassociate at a reaction temperature suitable for theenzymes and other materials in the reaction).

[0383] The first step of design and selection of the oligonucleotidesfor the INVADER oligonucleotide-directed cleavage is in accordance withthese sample general principles. Considered as sequence-specific probesindividually, each oligonucleotide may be selected according to theguidelines listed above. That is to say, each oligonucleotide willgenerally be long enough to be reasonably expected to hybridize only tothe intended target sequence within a complex sample, usually in the 20to 40 nucleotide range. Alternatively, because the INVADERoligonucleotide-directed cleavage assay depends upon the concertedaction of these oligonucleotides, the composite length of the 2oligonucleotides which span/bind to the X, Y, Z regions may be selectedto fall within this range, with each of the individual oligonucleotidesbeing in approximately the 13 to 17 nucleotide range. Such a designmight be employed if a non-thermostable cleavage means were employed inthe reaction, requiring the reactions to be conducted at a lowertemperature than that used when thermostable cleavage means areemployed. In some embodiments, it may be desirable to have theseoligonucleotides bind multiple times within a single target nucleic acid(e.g., to bind to multiple variants or multiple similar sequences withina target). It is not intended that the method of the present inventionbe limited to any particular size of the probe or INVADERoligonucleotide.

[0384] The second step of designing an oligonucleotide pair for thisassay is to choose the degree to which the upstream “INVADER”oligonucleotide sequence will overlap into the downstream “probe”oligonucleotide sequence, and consequently, the sizes into which theprobe will be cleaved. A key feature of this assay is that the probeoligonucleotide can be made to “turn over,” that is to say probe can bemade to depart to allow the binding and cleavage of other copies of theprobe molecule, without the requirements of thermal denaturation ordisplacement by polymerization. While in one embodiment of this assayprobe turnover may be facilitated by an exonucleolytic digestion by thecleavage agent, it is central to the present invention that the turnoverdoes not require this exonucleolytic activity. For example, in someembodiments, a reaction temperature and reaction conditions are selectedso as to create an equilibrium wherein the probe hybridizes anddisassociates from the target. In other embodiments, temperature andreaction conditions are selected so that unbound probe can initiatebinding to the target strand and physically displace bound probe. Instill other embodiments, temperature and reaction conditions areselected such that either or both mechanisms of probe replacement mayoccur in any proportion. The method of the present invention is notlimited to any particular mechanism of probe replacement. By anymechanism, when the probe is bound to the target to form a cleavagestructure, cleavage can occur. The continuous cycling of the probe onand off of the target allows multiple probes to bind and be cleaved foreach copy of a target nucleic acid.

[0385] i) Choosing the Amount of Sequence Overlap

[0386] One way of accomplishing such turnover, where the INVADERoligonucleotide and probe oligonucleotide share a region ofcomplementarity, can be envisioned by considering the diagram in FIG.29. It can be seen that the T_(m) of each oligonucleotide will be afunction of the full length of that oligonucleotide: i.e., the T_(m) ofthe INVADER oligonucleotide=T_(m)(Y+X), and the T_(m) of theprobe=T_(m)(X+Y) for the probe. When the probe is cleaved the X regionis released, leaving the Z section. If the T_(m) of Z is less than thereaction temperature, and the reaction temperature is less than theT_(m)(X+Z), then cleavage of the probe will lead to the departure of Z,thus allowing a new (X+Z) to hybridize. It can be seen from this examplethat the X region must be sufficiently long that the release of X willdrop the T_(m) of the remaining probe section below the reactiontemperature: a G-C rich X section may be much shorter than an A-T rich Xsection and still accomplish this stability shift.

[0387] In other embodiments described herein, probe turn over is notrelated to a change in T_(m) caused by cleavage of the probe, but ratheris related to the association and disassociation behavior of the probein the selected conditions, regardless of cleavage. Thus, it is notintended that the present invention be limited to the use of probesthat, upon cleavage, yield products having a T_(m)s below the reactiontemperature, as described above.

[0388] ii) Non-sequence Overlaps

[0389] It has been determined that the relationship between the 3′ endof the upstream oligonucleotide and the desired site of cleavage on theprobe should be carefully designed. It is known that the preferred siteof cleavage for the types of structure-specific endonucleases employedherein is one basepair into a duplex (Lyamichev et al., supra). It waspreviously believed that the presence of an upstream oligonucleotide orprimer allowed the cleavage site to be shifted away from this preferredsite, into the single stranded region of the 5′ arm (Lyamichev et al.,supra and U.S. Pat. No. 5,422,253). In contrast to this previouslyproposed mechanism, and while not limiting the present invention to anyparticular mechanism, it is believed that the nucleotide immediately 5′,or upstream of the cleavage site on the probe (including miniprobe andmid-range probes) should be able to basepair with the target forefficient cleavage to occur. In the case of the present invention, thiswould be the nucleotide in the probe sequence immediately upstream ofthe intended cleavage site. In addition, as described herein, it hasbeen observed that in order to direct cleavage to that same site in theprobe, the upstream oligonucleotide should have its 3′ base (i.e., nt)immediately upstream of the intended cleavage site of the probe. Inembodiments where the INVADER and probe oligonucleotides share asequence overlap, this places the 3′ terminal nucleotide of the upstreamoligonucleotide and the base of the probe oligonucleotide 5′ of thecleavage site in competition for pairing with the correspondingnucleotide of the target strand.

[0390] To examine the outcome of this competition (i.e. which base ispaired during a successful cleavage event), substitutions were made inthe probe and INVADER oligonucleotides such that either the probe or theINVADER oligonucleotide were mismatched with the target sequence at thisposition. The effects of both arrangements on the rates of cleavage wereexamined. When the INVADER oligonucleotide is unpaired at the 3′ end,the rate of cleavage was not reduced. If this base was removed, however,the cleavage site was shifted upstream of the intended site. Incontrast, if the probe oligonucleotide was not base-paired to the targetjust upstream of the site to which the INVADER oligonucleotide wasdirecting cleavage, the rate of cleavage was dramatically reduced,suggesting that when a competition exists, the probe oligonucleotide wasthe molecule to be base-paired in this position.

[0391] It appears that the 3′ end of the upstream INVADERoligonucleotide is unpaired during cleavage, and yet is important foraccurate positioning of the cleavage. To examine which part(s) of the 3′terminal nucleotide are required for the positioning of cleavage,INVADER oligonucleotides were designed that terminated on this end withnucleotides that were altered in a variety of ways. Sugars examinedincluded 2′ deoxyribose with a 3′ phosphate group, a dideoxyribose, 3′deoxyribose, 2′O-methyl ribose, arabinose and arabinose with a 3′phosphate. Abasic ribose, with and without 3′ phosphate were tested.Synthetic “universal” bases such at 3-nitropyrrole and 5-3 nitroindoleon ribose sugars were tested. Finally, a base-like aromatic ringstructure, acridine, linked to the 3′ end the previous nucleotidewithout a sugar group was tested. The results obtained support theconclusion that the aromatic ring of the base (at the 3′ end of theINVADER oligonuceotide) is an important moiety for accomplishing thedirection of cleavage to the desired site within the downstream probe.The 3′ terminal moiety of the INVADER oligonucleotide need not be a basethat is complementary to the target nucleic acid.

[0392] iii) Miniprobes and Mid-Range Probes;

[0393] As discussed above, the INVADER oligonucleotide-directed cleavageassay may be performed using INVADER and probe oligonucleotides thathave a length of about 13-25 nucleotides (typically 20-25 nucleotides).It is also contemplated that the oligonucleotides may themselves becomposed of shorter oligonucleotide sequences that align along a targetstrand but that are not covalently linked. This is to say that there isa nick in the sugar-phosphate backbone of the composite oligonucleotide,but that there is no disruption in the progression of base-pairednucleotides in the resulting duplex. When short strands of nucleic acidalign contiguously along a longer strand the hybridization of each isstabilized by the hybridization of the neighboring fragments because thebasepairs can stack along the helix as though the backbone was in factuninterrupted. This cooperativity of binding can give each segment astability of interaction in excess of what would be expected for thesegment hybridizing to the longer nucleic acid alone. One application ofthis observation has been to assemble primers for DNA sequencing,typically about 18 nucleotides long, from sets of three hexameroligonucleotides that are designed to hybridize in this way (Kotler etal. Proc. Natl. Acad. Sci. USA 90:4241 [1993]). The resultingdoubly-nicked primer can be extended enzymatically in reactionsperformed at temperatures that might be expected to disrupt thehybridization of hexamers, but not of 18-mers.

[0394] The use of composite or split oligonucleotides is applied withsuccess in the INVADER-directed cleavage assay. For example, the probeoligonucleotide may be split into two oligonucleotides that anneal in acontiguous and adjacent manner along a target oligonucleotide asdiagrammed in FIG. 57. In this Figure, the downstream oligonucleotide(analogous to the probe of FIG. 25) is assembled from two smallerpieces: a short segment of 6-10 nts (termed the “miniprobe”), that is tobe cleaved in the course of the detection reaction, and anoligonucleotide that hybridizes immediately downstream of the miniprobe(termed the “stacker”), that serves to stabilize the hybridization ofthe probe. To form the cleavage structure, an upstream oligonucleotide(the INVADER oligonucleotide) is provided to direct the cleavageactivity to the desired region of the miniprobe. Assembly of the probefrom non-linked pieces of nucleic acid (i.e., the miniprobe and thestacker) allows regions of sequences to be changed without requiring there-synthesis of the entire proven sequence, thus improving the cost andflexibility of the detection system. In addition, the use of unlinkedcomposite oligonucleotides makes the system more stringent in itsrequirement of perfectly matched hybridization to achieve signalgeneration, allowing this to be used as a sensitive means of detectingmutations or changes in the target nucleic acid sequences.

[0395] As illustrated in FIG. 57, in one embodiment, the methods of thepresent invention employ at least three oligonucleotides that interactwith a target nucleic acid to form a cleavage structure for astructure-specific nuclease. More specifically, the cleavage structurecomprises i) a target nucleic acid that may be either single-stranded ordouble-stranded (when a double-stranded target nucleic acid is employed,it may be rendered single-stranded, e.g., by heating); ii) a firstoligonucleotide, termed the “stacker,” that defines a first region ofthe target nucleic acid sequence by being the complement of that region(region W of the target as shown in FIG. 57); iii) a secondoligonucleotide, termed the “miniprobe,” that defines a second region ofthe target nucleic acid sequence by being the complement of that region(regions X and Z of the target as shown in FIG. 57); iv) a thirdoligonucleotide, termed the “INVADER,” the 5′ part of which defines athird region of the same target nucleic acid sequence (regions Y and Xin FIG. 57), adjacent to and downstream of the second target region(regions X and Z), and the second or 3′ part of which overlaps into theregion defined by the second oligonucleotide (region X depicts theregion of overlap). The resulting structure is diagrammed in FIG. 57. Asdescribed above for embodiments that do not employ a stacker, the regionshown as “X” can represent a region where there is a physical, but notsequence, overlap between the INVADER and probe oligonucleotides.

[0396] While not limiting the invention or the instant discussion to anyparticular mechanism of action, the diagram in FIG. 57 represents theeffect on the site of cleavage caused by this type of arrangement ofthree oligonucleotides. The design of these three oligonucleotides isdescribed below in detail. In FIG. 57, the 3′ ends of the nucleic acids(i.e., the target and the oligonucleotides) are indicated by the use ofthe arrowheads on the ends of the lines depicting the strands of thenucleic acids (and where space permits, these ends are also labeled“3′”). It is readily appreciated that the three oligonucleotides (theINVADER, the miniprobe and the stacker) are arranged in a parallelorientation relative to one another, while the target nucleic acidstrand is arranged in an anti-parallel orientation relative to the threeoligonucleotides. Further it is clear that the INVADER oligonucleotideis located upstream of the miniprobe oligonucleotide and that theminiprobe olignuceotide is located upstream of the stackeroligonucleotide and that with respect to the target nucleic acid strand,region W is upstream of region Z, region Z is upstream of upstream ofregion X and region X is upstream of region Y (that is region Y isdownstream of region X, region X is downstream of region Z and region Zis downstream of region W). Regions of complementarity between theopposing strands are indicated by the short vertical lines. While notintended to indicate the precise location of the site(s) of cleavage,the area to which the site of cleavage within the miniprobeoligonucleotide is shifted by the presence of the INVADERoligonucleotide is indicated by the solid vertical arrowhead. FIG. 57 isnot intended to represent the actual mechanism of action or physicalarrangement of the cleavage structure and further it is not intendedthat the method of the present invention be limited to any particularmechanism of action.

[0397] It can be considered that the binding of these oligonucleotidesdivides the target nucleic acid into four distinct regions: one regionthat has complementarity to only the stacker (shown as “W”); one regionthat has complementarity to only the miniprobe (shown as “Z”); oneregion that has complementarity only to the INVADER oligonucleotide(shown as “Y”); and one region that has complementarity to both theINVADER and miniprobe oligonucleotides (shown as “X”). As discussedabove, the INVADER oligonucleotide may also be employed such that aphysical overlap rather than a sequence overlap with the probe isprovided.

[0398] In addition to the benefits cited above, the use of a compositedesign for the oligonucleotides that form the cleavage structure allowsmore latitude in the design of the reaction conditions for performingthe INVADER-directed cleavage assay. When a longer probe (e.g., 16-25nt), as described above, is used for detection in reactions that areperformed at temperatures below the T_(m) of that probe, the cleavage ofthe probe may play a significant role in destabilizing the duplex ofwhich it is a part, thus allowing turnover and reuse of the recognitionsite on the target nucleic acid. In contrast, reaction temperatures thatare at or above the T_(m) of the probe mean that the probe molecules arehybridizing and releasing from the target quite rapidly even withoutcleavage of the probe. When an upstream INVADER oligonucleotide and acleavage means are provided the probe will be specifically cleaved, butthe cleavage will not be necessary to the turnover of the probe. When along probe (e.g., 16-25 nt) is used in this way the temperaturesrequired to achieve this state is high, around 65 to 70° C. for a 25-merof average base composition. Requiring the use of such elevatedtemperatures limits the choice of cleavage agents to those that are verythermostable, and may contribute to background in the reactions,depending of the means of detection, through thermal degradation of theprobe oligonucleotides. With miniprobes, this latter mechanism of probereplacement may be accomplished at a lower temperature. Thus, shorterprobes are preferred for embodiments using lower reaction temperatures.

[0399] The miniprobe of the present invention may vary in size dependingon the desired application. In one embodiment, the probe may berelatively short compared to a standard probe (e.g., 16-25 nt), in therange of 6 to 10 nucleotides. When such a short probe is used, reactionconditions can be chosen that prevent hybridization of the miniprobe inthe absence of the stacker oligonucleotide. In this way a short probecan be made to assume the statistical specificity and selectivity of alonger sequence. In the event of a perturbation in the cooperativebinding of the miniprobe and stacker nucleic acids, as might be causedby a mismatch within the short sequence that is otherwise complementaryto the target nucleic acid or at the junction between the contiguousduplexes, this cooperativity can be lost, dramatically reducing thestability of the shorter duplex (i.e., that of the miniprobe), and thusreducing the level of cleaved product in the assay of the presentinvention.

[0400] It is also contemplated that probes of intermediate size may beused. Such probes, in the 11 to 15 nucleotide range, may blend some ofthe features associated with the longer probes as originally described,these features including the ability to hybridize and be cleaved absentthe help of a stacker oligonucleotide. At temperatures below theexpected T_(m) of such probes, the mechanisms of turnover may be asdiscussed above for probes in the 20 nt range, and be dependent on theremoval of the sequence in the ‘X’ region for destabilization andcycling.

[0401] The mid-range probes may also be used at elevated temperatures,at or above their expected T_(m), to allow melting rather than cleavageto promote probe turnover. In contrast to the longer probes describedabove, however, the temperatures required to allow the use of such athermally driven turnover are much lower (about 40 to 60° C.), thuspreserving both the cleavage means and the nucleic acids in the reactionfrom thermal degradation. In this way, the mid-range probes may performin some instances like the miniprobes described above. In a furthersimilarity to the miniprobes, the accumulation of cleavage signal from amid-range probe may be helped under some reaction conditions by thepresence of a stacker.

[0402] To summarize, a standard long probe usually does not benefit fromthe presence of a stacker oligonucleotide downstream (the exceptionbeing cases where such an oligonucleotide may also disrupt structures inthe target nucleic acid that interfere with the probe binding), and itmay be used in conditions requiring several nucleotides to be removed toallow the oligonucleotide to release from the target efficiently. Iftemperature of the reaction is used to drive exchange of the probes,standard probes may require use of a temperature at which nucleic acidsand enzymes are at higher risk of thermal degradation.

[0403] The miniprobe is very short and performs optimally in thepresence of a downstream stacker oligonucleotide. The miniprobes arewell suited to reactions conditions that use the temperature of thereaction to drive rapid exchange of the probes on the target regardlessof whether any bases have been cleaved. In reactions with sufficientamount of the cleavage means, the probes that do bind will be rapidlycleaved before they melt off.

[0404] The mid-range or midiprobe combines features of these probes andcan be used in reactions like those favored by long probes, with longerregions of overlap (“X” regions) to drive probe turnover at lowertemperature. In a preferred embodiment, the midrange probes are used attemperatures sufficiently high that the probes are hybridizing to thetarget and releasing rapidly regardless of cleavage. The mid-range probemay have enhanced performance in the presence of a stacker under somecircumstances.

[0405] The distinctions between the mini-, midi- (i.e., mid-range) andlong probes are not contemplated to be inflexible and based only onlength. The performance of any given probe may vary with its specificsequence, the choice of solution conditions, the choice of temperatureand the selected cleavage means.

[0406] It is shown in Example 17 that the assemblage of oligonucleotidesthat comprises the cleavage structure of the present invention issensitive to mismatches between the probe and the target. The site ofthe mismatch used in Ex. 17 provides one example and is not intended tobe a limitation in location of a mismatch affecting cleavage. It is alsocontemplated that a mismatch between the INVADER oligonucleotide and thetarget may be used to distinguish related target sequences. In the3-oligonucleotide system, comprising an INVADER, a probe and a stackeroligonucleotide, it is contemplated that mismatches may be locatedwithin any of the regions of duplex formed between theseoligonucleotides and the target sequence. In a preferred embodiment, amismatch to be detected is located in the probe. In a particularlypreferred embodiment, the mismatch is in the probe, at the basepairimmediately upstream (i.e., 5′) of the site that is cleaved when theprobe is not mismatched to the target.

[0407] In another preferred embodiment, a mismatch to be detected islocated within the region ‘Z’ defined by the hybridization of aminiprobe. In a particularly preferred embodiment, the mismatch is inthe miniprobe, at the basepair immediately upstream (i.e., 5′) of thesite that is cleaved when the miniprobe is not mismatched to the target.

[0408] b) Design of the Reaction Conditions

[0409] Target nucleic acids that may be analyzed using the methods ofthe present invention that employ a 5′ nuclease or other appropriatecleavage agents include of both RNA and DNA. Such nucleic acids may beobtained using standard molecular biological techniques. For example,nucleic acids (RNA or DNA) may be isolated from a tissue sample (e.g., abiopsy specimen), tissue culture cells, samples containing bacteriaand/or viruses (including cultures of bacteria and/or viruses), etc. Thetarget nucleic acid may also be transcribed in vitro from a DNA templateor may be chemically synthesized or amplified in by polymerase chainreaction. Furthermore, nucleic acids may be isolated from an organism,either as genomic material or as a plasmid or similar extrachromosomalDNA, or they may be a fragment of such material generated by treatmentwith a restriction endonuclease or other cleavage agent, or a shearingforce, or it may be synthetic.

[0410] Assembly of the target, probe, and INVADER oligonucleotidenucleic acids into the cleavage reaction of the present invention usesprinciples commonly used in the design of oligonucleotide-basedenzymatic assays, such as dideoxynucleotide sequencing and polymerasechain reaction (PCR). As is done in these assays, the oligonucleotidesare provided in sufficient excess that the rate of hybridization to thetarget nucleic acid is very rapid. These assays are commonly performedwith 50 fmoles to 2 pmoles of each oligonucleotide per microliter ofreaction mixture, although they are not necessarily limited to thisrange In the Examples described herein, amounts of oligonucleotidesranging from 250 fmoles to 5 pmoles per microliter of reaction volumewere used. These values were chosen for the purpose of ease indemonstration and are not intended to limit the performance of thepresent invention to these concentrations. Other (e.g., lower)oligonucleotide concentrations commonly used in other molecularbiological reactions are also contemplated.

[0411] It is desirable that an INVADER oligonucleotide be immediatelyavailable to direct the cleavage of each probe oligonucleotide thathybridizes to a target nucleic acid. In some embodiments describedherein, the INVADER oligonucleotide is provided in excess over the probeoligonucleotide. While this is an effective means of making the INVADERoligonucleotide immediately available in such embodiments it is notintended that the practice of the present invention be limited toconditions wherein the INVADER oligonucleotide is in excess over theprobe, or to any particular ratio of INVADER-to-probe (e.g., in somepreferred embodiments described herein, the probe is provided in excessover the INVADER oligonucleotide). Another means of assuring thepresence of an INVADER oligonucleotide whenever a probe binds to atarget nucleic acid is to design the INVADER oligonucleotide tohybridize more stably to the target, i.e., to have a higher T_(m) thanthe probe. This can be accomplished by any of the means of increasingnucleic acid duplex stability discussed herein (e.g., by increasing theamount of complementarity to the target nucleic acid).

[0412] Buffer conditions should be chosen that will be compatible withboth the oligonucleotide/target hybridization and with the activity ofthe cleavage agent. The optimal buffer conditions for nucleic acidmodification enzymes, and particularly DNA modification enzymes,generally included enough mono- and di-valent salts to allow associationof nucleic acid strands by base-pairing. If the method of the presentinvention is performed using an enzymatic cleavage agent other thanthose specifically described here, the reactions may generally beperformed in any such buffer reported to be optimal for the nucleasefunction of the cleavage agent. In general, to test the utility of anycleavage agent in this method, test reactions are performed wherein thecleavage agent of interest is tested in the MOPS/MnCl₂/KCl buffer orMg-containing buffers described herein and in whatever buffer has beenreported to be suitable for use with that agent, in a manufacturer'sdata sheet, a journal article, or in personal communication.

[0413] The products of the INVADER oligonucleotide-directed cleavagereaction are fragments generated by structure-specific cleavage of theinput oligonucleotides. The resulting cleaved and/or uncleavedoligonucleotides may be analyzed and resolved by a number of methodsincluding, but not limited to, electrophoresis (on a variety of supportsincluding acrylamide or agarose gels, paper, etc.), chromatography,fluorescence polarization, mass spectrometry and chip hybridization. Insome Examples the invention is illustrated using electrophoreticseparation for the analysis of the products of the cleavage reactions.However, it is noted that the resolution of the cleavage products is notlimited to electrophoresis. Electrophoresis is chosen to illustrate themethod of the invention because electrophoresis is widely practiced inthe art and is easily accessible to the average practitioner. In otherExamples, the invention is illustrated without electrophoresis or anyother resolution of the cleavage products.

[0414] The probe and INVADER oligonucleotides may contain a label to aidin their detection following the cleavage reaction. The label may be aradioisotope (e.g., a ³²P or ³⁵S-labelled nucleotide) placed at eitherthe 5′ or 3′ end of the oligonucleotide or alternatively, the label maybe distributed throughout the oligonucleotide (i.e., a uniformly labeledoligonucleotide). The label may be a nonisotopic detectable moiety, suchas a fluorophore, that can be detected directly, or a reactive groupthat permits specific recognition by a secondary agent. For example,biotinylated oligonucleotides may be detected by probing with astreptavidin molecule that is coupled to an indicator (e.g., alkalinephosphatase or a fluorophore) or a hapten such as dioxigenin may bedetected using a specific antibody coupled to a similar indicator. Thereactive group may also be a specific configuration or sequence ofnucleotides that can bind or otherwise interact with a secondary agent,such as another nucleic acid, and enzyme, or an antibody.

[0415] c) Optimization of Reaction Conditions

[0416] The INVADER oligonucleotide-directed cleavage reaction is usefulto detect the presence of specific nucleic acids. In addition to theconsiderations listed above for the selection and design of the INVADERand probe oligonucleotides, the conditions under which the reaction isto be performed may be optimized for detection of a specific targetsequence.

[0417] One objective in optimizing the INVADER oligonucleotide-directedcleavage assay is to allow specific detection of the fewest copies of atarget nucleic acid. To achieve this end, it is desirable that thecombined elements of the reaction interact with the maximum efficiency,so that the rate of the reaction (e.g., the number of cleavage eventsper minute) is maximized. Elements contributing to the overallefficiency of the reaction include the rate of hybridization, the rateof cleavage, and the efficiency of the release of the cleaved probe.

[0418] The rate of cleavage will be a function of the cleavage meanschosen, and may be made optimal according to the manufacturer'sinstructions when using commercial preparations of enzymes or asdescribed in the examples herein. The other elements (rate ofhybridization, efficiency of release) depend upon the execution of thereaction, and optimization of these elements is discussed below.

[0419] Three elements of the cleavage reaction that significantly affectthe rate of nucleic acid hybridization are the concentration of thenucleic acids, the temperature at which the cleavage reaction isperformed and the concentration of salts and/or other charge-shieldingions in the reaction solution.

[0420] The concentrations at which oligonucleotide probes are used inassays of this type are well known in the art, and are discussed above.One example of a common approach to optimizing an oligonucleotideconcentration is to choose a starting amount of oligonucleotide forpilot tests; 0.01 to 2 μM is a concentration range used in manyoligonucleotide-based assays. When initial cleavage reactions areperformed, the following questions may be asked of the data: Is thereaction performed in the absence of the target nucleic acidsubstantially free of the cleavage product?; Is the site of cleavagespecifically positioned in accordance with the design of the INVADERoligonucleotide?; Is the specific cleavage product easily detected inthe presence of the uncleaved probe (or is the amount of uncut materialoverwhelming the chosen visualization method)?

[0421] A negative answer to any of these questions would suggest thatthe probe concentration is too high, and that a set of reactions usingserial dilutions of the probe should be performed until the appropriateamount is identified. Once identified for a given target nucleic acid ina give sample type (e.g., purified genomic DNA, body fluid extract,lysed bacterial extract), it should not need to be re-optimized. Thesample type is important because the complexity of the material presentmay influence the probe concentration optimum.

[0422] Conversely, if the chosen initial probe concentration is too low,the reaction may be slow, due to inefficient hybridization. Tests withincreasing quantities of the probe will identify the point at which theconcentration exceeds the optimum (e.g., at which it produces anundesirable effect, such as background cleavage not dependent on thetarget sequence, or interference with detection of the cleavedproducts). Since the hybridization will be facilitated by excess ofprobe, it is desirable, but not required, that the reaction be performedusing probe concentrations just below this point.

[0423] The concentration of INVADER oligonucleotide can be chosen basedon the design considerations discussed above. In some embodiments, theINVADER oligonucleotide is in excess of the probe oligonucleotide. In apreferred embodiment, the probe oligonucleotide is in excess of theINVADER oligonucleotide.

[0424] Temperature is also an important factor in the hybridization ofoligonucleotides. The range of temperature tested will depend in largepart on the design of the oligonucleotides, as discussed above. Where itis desired to have a reaction be run at a particular temperature (e.g.,because of an enzyme requirement, for convenience, for compatibilitywith assay or detection apparatuses, etc.), the oligonucleotides thatfunction in the reaction can be designed to optimally perform at thedesired reaction temperature. Each INVADER reaction includes at leasttwo target sequence-specific oligonucleotides for the primary reaction:an upstream INVADER oligonucleotide and a downstream probeoligonucleotide. In some preferred embodiments, the INVADERoligonucleotide is designed to bind stabily at the reaction temperature,while the probe is designed to freely associate and disassociate withthe target strand, with cleavage occurring only when an uncut probehybridizes adjacent to an overlapping INVADER oligonucleotide. Inpreferred embodiments, the probe includes a 5′ flap that is notcomplementary to the target, and this flap is released from the probewhen cleavage occurs. The released flap can be detected directly orindirectly. In some preferred embodiments, as discussed in detail below,the released flap participate as in INVADER oligonucleotide in asecondary reaction.

[0425] Optimum conditions for the INVADER assay are generally those thatallow specific detection of the smallest amount of a target nucleicacid. Such conditions may be characterized as those that yield thehighest target-dependent signal in a given timeframe, or for a givenamount of target nucleic acid, or that allow the highest rate of probecleavage (i.e., probes cleaved per minute). To select a probe sequencethat will perform optimally at a pre-selected reaction temperature, themelting temperature (T_(m)) of its analyte specific region (ASR, theregion that is complementary to the target nucleic acid) is calculatedusing the nearest-neighbor model and published parameters for DNA duplexformation (SantaLucia, J., Proc Natl Acad Sci U S A 95, 1460-5 (1998),Allawi, H. T. & SantaLucia, J., Jr. Biochemistry 36, 10581-94 (1997).However, there are several differences between the conditions underwhich the published parameters were measured and the conditions underwhich the INVADER assay is run in preferred embodiments. The saltconcentrations are often different than the solution conditions in whichthe nearest-neighbor parameters were obtained (1M NaCl and no divalentmetals). One can compensate for this factor by varying the valueprovided for the salt concentration within the melting temperaturecalculations. In addition to the salt concentration, the presence of andconcentration of the enzyme influences the optimal reaction temperature,and an additional adjustment should be made to the calculated T_(m) todetermine the optimal temperature at which to perform a reaction. Byobserving the optimal temperature for a number of INVADER reactions(i.e., the temperature at which the rate of signal accumulation ishighest) it has been possible to further alter the value for saltconcentration within these calculations to allow the algorithm for T_(m)calculation to be modified to instead provide an optimal cleavagereaction temperature for a given probe sequence. This additionaladjustment is termed a “salt correction”. As used herein, the term “saltcorrection” refers to a variation made in the value provided for a saltconcentration, for the purpose of reflecting the effect on a T_(m)calculation for a nucleic acid duplex of a non-salt parameter orcondition affecting said duplex. Variation of the values provided forthe strand concentrations will also affect the outcome of thesecalculations. By using a value of 0.5 M NaCl [SantaLucia, J., Proc NatlAcad Sci U S A 95, 1460-5 (1998)] and strand concentrations of about 1μM of the probe and 1 fM target, the algorithm used for calculatingprobe-target melting temperature has been adapted for use in predictingoptimal INVADER assay reaction temperature. For a set of about 30probes, the average deviation between optimal assay temperaturescalculated by this method and those experimentally determined was about1.5° C.

[0426] As noted above, the concentration of the cleavage agent canaffect the actual optimum temperature for a cleavage reaction.Additionally, different cleavage agents, even if used at identicalconcentrations, can affect reaction temperature optima differently(e.g., the difference between the calculated probe T_(m) and theobserved optimal reaction temperature may be greater for one enzyme thanfor another). Determination of appropriate salt corrections forreactions using different enzymes or concentrations of enzymes, or forany other variation made in reaction conditions, involves a two stepprocess of a) measuring reaction temperature optima under the newreaction conditions, and varying the salt concentration within the T_(m)algorithm to produce a calculated temperature matching or closelyapproximating the observed optima. Measurement of an optimum reactiontemperature generally involves performing reactions at a range oftemperatures selected such that the range allows observation of anincrease in performance as an optimal temperature is approached (eitherby increasing or decreasing temperatures), and a decrease in performancewhen an optimal temperature has been passed, thereby allowingidentification of the optimal temperature or temperature range [see, forexample, V. I. Lyamichev, et al., Biochemistry 39, No. 31: 9523-9532(2000)].

[0427] The length of the downstream probe analyte-specific region (ASR)is defined by the temperature selected for running the reaction, e.g.,63° C. in the experiments described in Examples 54 through 60. To selecta probe sequence based on a desired reaction temperature, the probesequence is selected in the following way (as illustrated for the designof a probe for the detection of a sequence difference at a particularlocation). Starting from the position of the variant nucleotide on thetarget DNA (position N, FIG. 112); the target base that is paired to theprobe nucleotide 5′ of the intended cleavage site), an iterativeprocedure is used by which the length of the ASR is increased by onebase pair until a calculated optimal reaction temperature (T_(m) plussalt correction to compensate for enzyme and any other reactionconditions effects) matching the desired reaction temperature isreached. The non-complementary arm of the probe is preferably selected(by a similar iterative process) to allow the secondary reaction tocycle at the same reaction temperature, and the entire probe design (ASRand 5′ noncomplementary arm) is screened using programs such as mfold[Zuker, M. Science 244, 48-52 (1989)] or Oligo 5.0 [Rychlik, W. &Rhoads, R. E. Nucleic Acids Res 17, 8543-51 (1989)] for the possibleformation of dimer complexes or secondary structures that couldinterfere with the reaction. The same principles are also followed forINVADER oligonucleotide design. The following describes design of anINVADER assay embodiment wherein the 3′ end of the INVADERoligonucleotide, at position N on the target DNA, is designed to have anucleotide not complementary to either allele suspected of beingcontained in the sample to be tested. The mismatch does not adverselyaffect cleavage [Lyamichev, V. et al. Nature Biotechnology 17, 292-296(1999)], and it can enhance probe cycling, presumably by minimizingcoaxial stabilization effects between the two probes. Briefly, startingfrom the position N, additional residues complementary to the target DNAstarting from residue N-1 are then added in the upstream direction untilthe stability of the INVADER-target hybrid exceeds that of the probe(and therefore the planned assay reaction temperature). In preferredembodiments, the stability of the INVADER-target hybrid exceeds that ofthe probe by 15-20° C.

[0428] In some embodiments, where the released cleavage fragment from aprimary reaction is to be used in a secondary reaction, one should alsoconsider the reaction conditions of the secondary reaction in designingthe oligonucleotides for the primary reaction (e.g., the sequence of thereleased non-complementary 5′ flap of the probe in the primary reactioncan be designed to optimally function in a secondary reaction). Forexample, as described in detail below, in some embodiments, a secondaryreaction is used where the released cleavage fragment from a primaryreaction hybridizes to a synthetic cassette to form a secondary cleavagereaction. In some preferred embodiments, the cassette comprises afluorescing moiety and a quenching moiety, wherein cleavage of thesecondary cleavage structure separates the fluorescing moiety from thequenching moiety, resulting in a detectable signal (e.g., FRETdetection). The secondary reaction can be configured a number ofdifferent ways. For example, in some embodiments, the synthetic cassettecomprises two oligonucleotides: an oligonucleotide that contains theFRET moieties and a FRET/INVADER oligonucleotide bridgingoligonucleotide that allows the INVADER oligonucleotide (i.e., thereleased flap from the primary reaction) and the FRET oligonucleotide tohybridize thereto, such that a cleavage structure is formed. In someembodiments, the synthetic cassette is provided as a singleoligonucleotide, comprising a hairpin structure (i.e., the FREToligonucleotide is connected at its 3′ end to the bridgingoligonucleotide by a loop). The loop may be nucleic acid, (e.g., astring of nucleotides, such as the four T residues depicted in severalFigures, including 113A) or a non-nucleic acid spacer or linker. Thelinked molecules may together be described as a FRET cassette. In thesecondary reaction using a FRET cassette the released flap from theprimary reaction, which acts as an INVADER oligonucleotide, should beable to associate and disassociate with the FRET cassette freely, sothat one released flap can direct the cleavage of multiple FRETcassettes. It is one aspect of the assay design that all of the probesequences may be selected to allow the primary and secondary reactionsto occur at the same optimal temperature, so that the reaction steps canrun simultaneously. In an alternative embodiment, the probes may bedesigned to operate at different optimal temperatures, so that thereactions steps are not simultaneously at their temperature optima. Asnoted above, the same iterative process used to select the ASR of theprobe can be used in the design of the portion of the primary probe thatparticipates in a secondary reaction.

[0429] Another determinant of hybridization efficiency is the saltconcentration of the reaction. In large part, the choice of solutionconditions will depend on the requirements of the cleavage agent, andfor reagents obtained commercially, the manufacturer's instructions area resource for this information. When developing an assay utilizing anyparticular cleavage agent, the oligonucleotide and temperatureoptimizations described above should be performed in the bufferconditions best suited to that cleavage agent.

[0430] A “no enzyme” control allows the assessment of the stability ofthe labeled oligonucleotides under particular reaction conditions, or inthe presence of the sample to be tested e.g., in assessing the samplefor contaminating nucleases). In this manner, the substrate andoligonucleotides are placed in a tube containing all reactioncomponents, except the enzyme and treated the same as theenzyme-containing reactions. Other controls may also be included. Forexample, a reaction with all of the components except the target nucleicacid will serve to confirm the dependence of the cleavage on thepresence of the target sequence.

[0431] d) Selection of a Cleavage Agent

[0432] As demonstrated in a number of the Examples, some 5′ nucleases donot require an upstream oligonucleotide to be active in a cleavagereaction. Although cleavage may be slower without the upstreamoligonucleotide, it may still occur (Lyamichev et al., Science 260:778[1993], Kaiser et al., J. Biol. Chem., 274:21387 [1999]). When a DNAstrand is the template or target strand to which probe oligonucleotidesare hybridized, the 5′ nucleases derived from DNA polymerases and someflap endonucleases (FENs), such as that from Methanococcus jannaschii,can cleave quite well without an upstream oligonucleotide providing anoverlap (Lyamichev et al., Science 260:778 [1993], Kaiser et al., J.Biol. Chem., 274:21387 [1999], and U.S. Pat. No. 5,843,669, hereinincorporated by reference in its entirety). These nucleases may beselected for use in some embodiments of the INVADER assay, e.g., inembodiments wherein cleavage of th probe in the absence of an INVADERoligonucleotide gives a different cleavage product, which does notinterfere with the intended analysis, or wherein both types of cleavage,INVADER oligonucleotide-directed and INVADERoligonucleotide-independent, are intended to occur.

[0433] In other embodiments it is preferred that cleavage of the probebe dependent on the presence of an upstream INVADER oligonucleotide, andenzyme having this requirement would be used. Other FENs, such as thosefrom Archeaoglobus fulgidus (Afu) and Pyrococcus furiosus (Pfu), cleavean overlapped structure on a DNA target at so much greater a rate thanthey do a non-overlapping structure (i.e., either missing the upstreamoligonucleotide or having a non-overlapping upstream oligonucleotide)that they can be viewed as having an essentially absolute requirementfor the overlap (Lyamichev et al., Nat. Biotechnol., 17:292 [1999],Kaiser et al., J. Biol. Chem., 274:21387 [1999]). When an RNA target ishybridized to DNA oligonucleotide probes to form a cleavage structure,many FENs cleave the downstream DNA probe poorly, regardless of thepresence of an overlap. On such an RNA-containing structure, the 5′nucleases derived from DNA polymerases have a strong requirement for theoverlap, and are essentially inactive in its absence.

[0434] e) Probing for Multiple Alleles

[0435] The INVADER oligonucleotide-directed cleavage reaction is alsouseful in the detection and quantification of individual variants oralleles in a mixed sample population. By way of example, such a needexists in the analysis of tumor material for mutations in genesassociated with cancers. Biopsy material from a tumor can have asignificant complement of normal cells, so it is desirable to detectmutations even when present in fewer than 5% of the copies of the targetnucleic acid in a sample. In this case, it is also desirable to measurewhat fraction of the population carries the mutation. Similar analysesmay also be done to examine allelic variation in other gene systems, andit is not intended that the method of the present invention by limitedto the analysis of tumors.

[0436] As demonstrated below, in one embodiment, reactions can beperformed under conditions that prevent the cleavage of probes bearingeven a single-nucleotide difference mismatch within the region of thetarget nucleic acid termed “Z” in FIG. 29, but that permit cleavage of asimilar probe that is completely complementary to the target in thisregion. In a preferred embodiment, a mismatch is positioned at thenucleotide in the probe that is 5′ of the site where cleavage occurs inthe absence of the mismatch.

[0437] In other embodiments, the INVADER assay may be performed underconditions that have a tight requirement for an overlap (e.g., using theAfu FEN for DNA target detection or the 5′ nuclease of DNA polymerasefor RNA target detection, as described above), providing an alternativemeans of detecting single nucleotide or other sequence variations. Inone embodiment, the probe is selected such that the target basesuspected of varying is positioned at the 5′ end of thetarget-complementary region of this probe. The upstream INVADERoligonucleotide is positioned to provide a single base of overlap. Ifthe target and the probe oligonucleotide are complementary at the basein question, the overlap forms and cleavage can occur. This embodimentis diagrammed in FIG. 112. However, if the target does not complementthe probe at this position, that base in the probe becomes part of anon-complementary 5′ arm, no overlap between the INVADER oligonucleotideand probe oligonucleotide exists, and cleavage is suppressed.

[0438] It is also contemplated that different sequences may be detectedin a single reaction. Probes specific for the different sequences may bedifferently labeled. For example, the probes may have different dyes orother detectable moieties, different lengths, or they may havedifferences in net charges of the products after cleavage. Whendifferently labeled in one of these ways, the contribution of eachspecific target sequence to final product can be tallied. This hasapplication in detecting the quantities of different versions of a genewithin a mixture. Different genes in a mixture to be detected andquantified may be wild type and mutant genes (e.g., as may be found in atumor sample, such as a biopsy). In this embodiment, one might designthe probes to precisely the same site, but one to match the wild-typesequence and one to match the mutant. Quantitative detection of theproducts of cleavage from a reaction performed for a set amount of timewill reveal the ratio of the two genes in the mixture. Such analysis mayalso be performed on unrelated genes in a mixture. This type of analysisis not intended to be limited to two genes. Many variants within amixture may be similarly measured.

[0439] Alternatively, different sites on a single gene may be monitoredand quantified to verify the measurement of that gene. In thisembodiment, the signal from each probe would be expected to be the same.

[0440] It is also contemplated that multiple probes may be used that arenot differently labeled, such that the aggregate signal is measured.This may be desirable when using many probes designed to detect a singlegene to boost the signal from that gene. This configuration may also beused for detecting unrelated sequences within a mix. For example, inblood banking it is desirable to know if any one of a host of infectiousagents is present in a sample of blood. Because the blood is discardedregardless of which agent is present, different signals on the probeswould not be required in such an application of the present invention,and may actually be undesirable for reasons of confidentiality.

[0441] Just as described for the two-oligonucleotide system, above, thespecificity of the detection reaction will be influenced by theaggregate length of the target nucleic acid sequences involved in thehybridization of the complete set of the detection oligonucleotides. Forexample, there may be applications in which it is desirable to detect asingle region within a complex genome. In such a case the set ofoligonucleotides may be chosen to require accurate recognition byhybridization of a longer segment of a target nucleic acid, often in therange of 20 to 40 nucleotides. In other instances it may be desirable tohave the set of oligonucleotides interact with multiple sites within atarget sample. In these cases one approach would be to use a set ofoligonucleotides that recognize a smaller, and thus statistically morecommon, segment of target nucleic acid sequence.

[0442] In one preferred embodiment, the INVADER and stackeroligonucleotides may be designed to be maximally stable, so that theywill remain bound to the target sequence for extended periods during thereaction. This may be accomplished through any one of a number ofmeasures well known to those skilled in the art, such as adding extrahybridizing sequences to the length of the oligonucleotide (up to about50 nts in total length), or by using residues with reduced negativecharge, such as phosphorothioates or peptide-nucleic acid residues, sothat the complementary strands do not repel each other to degree thatnatural strands do. Such modifications may also serve to make theseflanking oligonucleotides resistant to contaminating nucleases, thusfurther ensuring their continued presence on the target strand duringthe course of the reaction. In addition, the INVADER and stackeroligonucleotides may be covalently attached to the target (e.g., throughthe use of psoralen cross-linking).

[0443] II. Effect of ARRESTOR Molecules on Signal and Background inSequential Invasive Cleavage Reactions.

[0444] As described above, and demonstrated in Example 36, theconcentration of the probe that is cleaved can be used to increase therate of signal accumulation, with higher concentrations of probeyielding higher final signal. However, the presence of large amounts ofresidual uncleaved probe can present problems for subsequent use of thecleaved products for detection or for further amplification. If thesubsequent step is a simple detection (e.g., by gel resolution), theexcess uncut material may cause background by streaking or scattering ofsignal, or by overwhelming a detector (e.g., over-exposing a film in thecase of radioactivity, or exceeding the quantitative detection limits ofa fluorescence imager). This can be overcome by partitioning the productfrom the uncut probe (e.g., by using the charge reversal methoddescribed in Example 22 and discussed in detail below). In more complexdetection methods, the cleaved product may be intended to interact withanother entity to indicate cleavage. As noted above, the cleaved productcan be used in any reaction that makes use of oligonucleotides, such ashybridization, primer extension, ligation, or the direction of invasivecleavage. In each of these cases, the fate of the residual uncut probeshould be considered in the design of the reaction. In a primerextension reaction, the uncut probe can hybridize to a template forextension. If cleavage is required to reveal the correct 3′ end forextension, the hybridized uncut probe will not be extended. It may,however, compete with the cleaved product for the template. If thetemplate is in excess of the combination of cleaved and uncleaved probe,then both of the latter should be able to find a copy of template forbinding. If, however, the template is limiting, any competition mayreduce the portion of the cleaved probe that can find successfully bindto the available template. If a vast excess of probe was used to drivethe initial reaction, the remainder may also be in vast excess over thecleavage product, and thus may provide a very effective competitor,thereby reducing the amount of the final reaction (e.g., extension)product for ultimate detection.

[0445] The participation of the uncut probe material in a secondaryreaction can also contribute to background in these reactions. While thepresentation of a cleaved probe for a subsequent reaction may representan ideal substrate for the enzyme to be used in the next step, someenzymes may also be able to act, albeit inefficiently, on the uncutprobe as well. It was shown in Example 43 that transcription can bepromoted from a nicked promoter even when one side of the nick hasadditional unpaired nucleotides (termed a “branched promoter” in thatExample). Similarly, when the subsequent reaction is to be an invasivecleavage, the uncleaved probe may bind to the elements intended to formthe second cleavage structure with the cleaved probe. Two of thepossible configurations are shown schematically in FIGS. 105 and 106.The right hand structure in the second step in each Figure shows apossible configuration formed by the secondary reaction elements (e.g.,secondary targets and/or probes) and the uncleaved primary probe. Ineach of these cases, it was found that some of the 5′ nucleasesdescribed herein can catalyze some measure of cleavage of thesedefective structures. Even at a low level, this aberrant cleavage can bemisinterpreted as positive target-specific cleavage signal.

[0446] With these negative effects of the surfeit of uncut probeconsidered, there is clearly a need for some method of preventing theseinteractions. As noted above, it is possible to partition the cleavedproduct from the uncut probe after the primary reaction by traditionalmethods. However, these methods are often time consuming, may beexpensive (e.g., disposable columns, gels, etc.), and may increase therisk for sample mishandling or contamination. It is far preferable toconfigure the sequential reactions such that the original sample neednot be removed to a new vessel for subsequent reaction.

[0447] The present invention provides a method for reducing interactionsbetween the primary probe and other reactants. This method provides ameans of specifically diverting the uncleaved probes from participationin the subsequent reactions. The diversion is accomplished by theinclusion in the next reaction step an agent designed to specificallyinteract with the uncleaved primary probe. While the primary probe in aninvasive cleavage reaction is discussed for reasons of convenience, itis contemplated that the ARRESTOR molecules may be used at any reactionstep within a chain of invasive cleavage steps, as needed or desired forthe design of an assay. It is not intended that the ARRESTOR moleculesof the present invention be limited to any particular step.

[0448] The method of diverting the residual uncut probes from a primaryreaction makes use of agents that can be specifically designed orselected to bind to the uncleaved probe molecules with greater affinitythan to the cleaved probes, thereby allowing the cleaved probe speciesto effectively compete for the elements of the subsequent reaction, evenwhen the uncut probe is present in vast excess. These agents have beentermed “ARRESTOR molecules,” due to their function of stopping orarresting the primary probe from participation in the later reaction. Invarious Examples below, an oligonucleotide is provided as an ARRESTORoligonucleotide in an invasive cleavage assay. It can be appreciatedthat any molecule or chemical that can discriminate between thefull-length uncut probe and the cleaved probe, and that can bind orotherwise disable the uncleaved probe preferentially may be configuredto act as an ARRESTOR molecules within the meaning of the presentinvention. For example, antibodies can be derived with such specificity,as can the “aptamers” that can be selected through multiple steps of invitro amplification (e.g., “SELEX,” U.S. Pat. Nos. 5,270,163 and5,567,588; herein incorporated by reference) and specific rounds ofcapture or other selection means.

[0449] In one embodiment, the ARRESTOR molecule is an oligonucleotide.In another embodiment the ARRESTOR oligonucleotides is a compositeoligonucleotide, comprising two or more short oligonucleotides that arenot covalently linked, but that bind cooperatively and are stabilized byco-axial stacking. In a preferred embodiment, the oligonucleotide ismodified to reduce interactions with the cleavage agents of the presentinvention. When an oligonucleotide is used as an ARRESTORoligonucleotide, it is intended that it not participate in thesubsequent reactive step. Consideration of the schematic diagrams inFIGS. 105 and 106, particularly the right-most Figure in step 2 b ofeach Figure, will show that the binding of the ARRESTOR oligonucleotideto the primary probe may, either with the participation of the secondarytarget, or without such participation, create a bifurcated structurethat is a substrate for cleavage by the 5′ nucleases used in someembodiments of the methods of the present invention. Formation of suchstructures would lead to some level of unintended cleavage that couldcontribute to background, reduce specific signal or compete for theenzyme. It is preferable to provide ARRESTOR oligonucleotides that willnot create such cleavage structures. One method of doing this is to addto the ARRESTOR oligonucleotides such modifications as have been foundto reduce the activity of INVADER oligonucleotides, as the INVADERoligonucleotides occupy a similar position within a cleavage structure(i.e., the 3′ end of the INVADER oligonucleotide positions the site ofcleavage of an unpaired 5′ arm). Modification of the 3′ end of theINVADER oligonucleotides was examined for the effects on cleavage inExample 35; a number of the modifications tested were found to besignificantly debilitating to the function of the INVADERoligonucleotide. Other modifications not described herein may be easilycharacterized by performing such a test using the cleavage enzyme to beused in the reaction for which the ARRESTOR oligonucleotide is intended.

[0450] In a preferred embodiment, the backbone of an ARRESTORoligonucleotide is modified. This may be done to increase the resistanceto degradation by nucleases or temperature, or to provide duplexstructure that is a less favorable substrate for the enzyme to be used(e.g., A-form duplex vs. B-form duplex). In particularly preferredembodiment, the backbone modified oligonucleotide further comprises a 3′terminal modification. In a preferred embodiment, the modificationscomprise 2′O-methyl substitution of the nucleic acid backbone, while ina particularly preferred embodiment, the 2′O-methyl modifiedoligonucleotide further comprises a 3′ terminal amine group.

[0451] The purpose of the ARRESTOR oligonucleotide is to allow theminority population of cleaved probe to effectively compete with theuncleaved probe for binding whatever elements are to be used in the nextstep. While an ARRESTOR oligonucleotide that can discriminate betweenthe two probe species absolutely (i.e., binding only to uncut and neverto cut) may be of the greatest benefit in some embodiments, it isenvisioned that in many applications, including the sequential INVADERassays described herein, the ARRESTOR oligonucleotide of the presentinvention may perform the intended function with only partialdiscrimination. When the ARRESTOR oligonucleotide has some interactionwith the cleaved probe, it may prevent detection of some portion ofthese cleavage products, thereby reducing the absolute level of signalgenerated from a given amount of target material. If this same ARRESTORoligonucleotide has the simultaneous effect of reducing the backgroundof the reaction (i.e., from non-target specific cleavage) by a factorthat is greater than the factor of reduction in the specific signal,then the significance of the signal (i.e., the ratio of signal tobackground), is increased, even with the lower amount of absolutesignal. Any potential ARRESTOR molecule design may be tested in a simplefashion by comparing the levels of background and specific signals fromreactions that lack ARRESTOR molecules to the levels of background andspecific signal from similar reactions that include ARRESTORoligonucleotides. Each of the reactions described in Examples 49-53demonstrate the use of such comparisons, and these can easily be adaptedby those skilled in the art to other ARRESTOR molecules and targetembodiments. What constitutes an acceptable level of tradeoff ofabsolute signal for specificity will vary for different applications(e.g., target levels, read-out sensitivity, etc.), and can be determinedby any individual user using the methods of the present invention.

[0452] III. Signal Enhancement by Incorporating The Products of anInvasive Cleavage Reaction Into a Subsequent Invasive Cleavage Reaction

[0453] As noted above, the oligonucleotide product released by theinvasive cleavage can be used subsequently in any reaction or read-outmethod that uses oligonucleotides in the size range of a cleavageproduct. In addition to the reactions involving primer extension andtranscription, described herein, another enzymatic reaction that makesuse of oligonucleotides is the invasive cleavage reaction. The presentinvention provide means of using the oligonucleotide released in aprimary invasive cleavage reaction as a component to complete a cleavagestructure to enable a secondary invasive cleavage reaction. One possibleconfiguration of a primary cleavage reaction supplying a component for asecondary cleavage structure is diagrammed in FIG. 96. Is not intendedthat the sequential use of the invasive cleavage product be limited to asingle additional step. It is contemplated that many distinct invasivecleavage reactions may be performed in sequence.

[0454] The polymerase chain reaction uses a DNA replication method tocreate copies of a targeted segment of nucleic acid at a logarithmicrate of accumulation. This is made possible by the fact that when thestrands of DNA are separated, each individual strand contains sufficientinformation to allow assembly of a new complementary strand. When thenew strands are synthesized the number of identical molecules hasdoubled. Within 20 iterations of this process, the original may becopied 1 million-fold, making very rare sequences easily detectable. Themathematical power of a doubling reaction has been incorporated into anumber of amplification assays, several of which are cited in Table 1.

[0455] By performing multiple, sequential invasive cleavage reactionsthe method of the present invention captures an exponential mathematicaladvantage without producing additional copies of the target analyte. Ina simple invasive cleavage reaction the yield, Y, is simply the turnoverrate, K, multiplied by the time of the reaction, t (i.e., Y=(K)(t)). IfY is used to represent the yield of a simple reaction, then the yield ofa compound (i.e., a multiple, sequential reaction), assuming that eachof the individual invasive cleavage steps has the same turnover rate,can be simply represented as Y^(n), where n is the number of invasivecleavage reactions that have been performed in the series. If the yieldsof each step differ the ultimate yield can be represented as the productof the multiplication of the yields of each individual reaction in theseries. For example, if a primary invasive cleavage reaction can produceone thousand products in 30 minutes, and each of those products can inturn participate in 1000 additional reactions, there will be 1000²copies (1000×1000) of the ultimate product in a second reaction. If athird reaction is added to the series, then the theoretical yield willbe 1000³ (1000×1000×1000). In the methods of the present invention theexponent comes from the number of invasive cleavage reactions in thecascade. This can be contrasted to the amplification methods describedabove (e.g., PCR) in which Y is limited to 2 by the number of strands induplex DNA, and the exponent n is the number of cycles performed, sothat many iterations are necessary to accumulate large amounts ofproduct.

[0456] To distinguish the exponential amplifications described abovefrom those of the present invention, the former can be consideredreciprocating reactions because the products the reaction feed back intothe same reaction (e.g., event one leads to some number of events 2, andeach event 2 leads back to some number of events 1). In contrast, theevents of the present invention are sequential (e.g., event 1 leads tosome number of events 2; each event 2 leads to some number of events 3,etc., and no event can contribute to an event earlier in the chain).

[0457] The sensitivity of the reciprocating methods is also one of thegreatest weaknesses when these assays are used to determine if a targetnucleic acid sequence is present or absent in a sample. Because theproduct of these reactions is detectable copies of the startingmaterial, contamination of a new reaction with the products of anearlier reaction can lead to false positive results, (i.e., the apparentdetection of the target nucleic acid in samples that do not actuallycontain any of that target analyte). Furthermore, because theconcentration of the product in each positive reaction is so high,amounts of DNA sufficient to create a strong false positive signal canbe communicated to new reactions very easily either by contact withcontaminated instruments or by aerosol. In contrast to the reciprocatingmethods, the most concentrated product of the sequential reaction (i.e.,the product released in the ultimate invasive cleavage event) is notcapable of initiating a like reaction or cascade if carried over to afresh test sample. This is a marked advantage over the exponentialamplification methods described above because the reactions of thepresent invention may be performed without the costly containmentarrangements (e.g., either by specialized instruments or by separatelaboratory space) required by any reciprocating reaction. While theproducts of a penultimate event may be inadvertently transferred toproduce a background of the ultimate product in the absence of the atarget analyte, the contamination would need to be of much greatervolume to give an equivalent risk of a false positive result.

[0458] When the term sequential is used it is not intended to limit theinvention to configurations in which that one invasive cleavage reactionor assay must be completed before the initiation of a subsequentreaction for invasive cleavage of a different probe. Rather, the termrefers to the order of events as would occur if only single copies ofeach of the oligonucleotide species were used in an assay. The primaryinvasive cleavage reaction refers to that which occurs first, inresponse to the formation of the cleavage structure on the targetnucleic acid. Subsequent reactions may be referred to as secondary,tertiary and so forth, and may involve artificial “target” strands thatserve only to support assembly of a cleavage structure, and which areunrelated to the nucleic acid analyte of interest. While the completeassay may, if desired, be configured with each step of invasive cleavageseparated either in space (e.g., in different reaction vessels) or intime (e.g., using a shift in reaction conditions, such as temperature,enzyme identity or solution condition, to enable the later cleavageevents), it is also contemplated that all of the reaction components maybe mixed so that secondary reactions may be initiated as soon as productfrom a primary cleavage becomes available. In such a format, primary,secondary and subsequent cleavage events involving different copies ofthe cleavage structures may take place simultaneously.

[0459] Several levels of this sort of linear amplification can beenvisioned, in which each successive round of cleavage produces anoligonucleotide that can participate in the cleavage of a differentprobe in subsequent rounds. The primary reaction would be specific forthe analyte of interest with secondary (and tertiary, etc.) reactionsbeing used to generate signal while still being dependent on the primaryreaction for initiation.

[0460] The released product may perform in several capacities in thesubsequent reactions. One of the possible variations is shown in FIG.96, in which the product of one invasive cleavage reaction becomes theINVADER oligonucleotide to direct the specific cleavage of another probein a second reaction. In FIG. 96, the first invasive cleavage structureis formed by the annealing of the INVADER oligonucleotide (“Invader”)and the probe oligonucleotide (“Probe 1”) to the first target nucleicacid (“Target 1”). The target nucleic acid is divided into three regionsbased upon which portions of the INVADER and probe oligonucleotides arecapable of hybridizing to the target (as discussed above and as shown inFIG. 25). Region 1 (region Y in FIG. 25) of the target hascomplementarity to only the INVADER oligonucleotide; region 3 (region Zin FIG. 25) of the target has complementarity to only the probe; andregion 2 (region X in FIG. 25) of the target has complementarity to boththe INVADER and probe oligonucleotides. It is noted that the sequentialinvasive cleavage reaction diagrammed in FIG. 96 employs an INVADER anda probe oligonucleotide; the sequential cleavage reaction is not limitedto the use of such a first cleavage structure. The first cleavagestructure in the sequential reaction may also employ an INVADERoligonucleotide, a mini probe and a stacker oligonucleotide as discussedabove. Further, as discussed above, the overlap in any or all of thecleavage structures in the sequential reactions may comprise moietiesother than overlapping complementary bases, such that the region shownas “X” represents a region where there is a physical rather thansequence overlap between the INVADER and probe oligonucleotides

[0461] In FIG. 96, cleavage of Probe 1 releases the “Cut Probe 1”(indicated by the hatched line in both the cleaved and uncleaved Probe 1in FIG. 96). The released Probe 1 is then used as the INVADERoligonucleotide in second cleavage. The second cleavage structure isformed by the annealing of the Cut Probe 1, a second probeoligonucleotide (“Probe 2”) and a second target nucleic acid (“Target2”) In some embodiments, Probe 2 and the second target nucleic acid arecovalently connected, preferably at their 3′ and 5′ ends, respectively,thus forming a hairpin stem and loop, termed herein a “cassette”. Theloop may be nucleic acid, (e.g., a string of nucleotides, such as thefour T residues depicted in several Figures, including 113A) or anon-nucleic acid spacer or linker. Inclusion of an excess of thecassette molecule allows each Cut Probe 1 to serve as an INVADER todirect the cleavage of multiple copies of the cassette.

[0462] Probe 2 may be labeled (e.g., as indicated by the star in FIG.96) and detection of cleavage of the second cleavage structure may beaccomplished by detecting the labeled cut Probe 2; the label may aradioisotope (e.g.. ³²P, 35S), a fluorophore (e.g., fluorescein), areactive group capable of detection by a secondary agent (e.g.,biotin/streptavidin), a positively charged adduct which permitsdetection by selective charge reversal (as discussed in Section IVabove), etc. Alternatively, the cut Probe 2 may used in a tailingreaction, or to complete or activate a protein binding site, or may bedetected or used by any of the means for detecting or using anoligonucleotide described herein.

[0463] Another possible configuration for performing a sequentialinvasive cleavage reaction is diagrammed in FIG. 97. In this embodiment,probe oligonucleotides that are cleaved in the primary reaction can bedesigned to fold back on themselves (i.e., they contain a region ofself-complementarity) to create a molecule that can serve as both theINVADER and target oligonucleotide (termed here an “IT” complex). The ITcomplex then enables cleavage of a different probe present in thesecondary reaction. Inclusion of an excess of the secondary probemolecule (“Probe 2”), allows each IT molecule to serve as the platformfor the generation of multiple copies of cleaved secondary probe. InFIG. 97, the regions of self-complementarity contained within the 5′portion of the INVADER oligonucleotide is indicated by the hatchedovals; the arrow between these two ovals indicates that these tworegions can self-pair (as shown in the “Cut Probe 1”). The targetnucleic acid is divided into three regions based upon which portions ofthe INVADER and probe oligonucleotides are capable of hybridizing to thetarget (as discussed above and it is noted that the target may bedivided into four regions if a stacker oligonucleotide is employed). Thesecond cleavage structure is formed by the annealing of the second probe(“Probe 2”) to the fragment of Probe 1 (“Cut Probe 1 ”) that wasreleased by cleavage of the first cleavage structure. The Cut Probe 1forms a hairpin or stem/loop structure near its 3′ terminus by virtue ofthe annealing of the regions of self-complementarity contained withinCut Probe 1 (this self-annealed Cut Probe 1 forms the IT complex). TheIT complex (Cut Probe 1) is divided into three regions. Region 1 of theIT complex has complementarity to the 3′ portion of Probe 2; region 2has complementarity to both the 3′ end of Cut Probe 1 and to the 5′portion of Probe 2 (analogous to the region of overlap “X” shown in FIG.25); and region 3 contains the region of self-complementarity (i.e.,region 3 is complementary to the 3′ portion of the Cut Probe 1). Notethat with regard to the IT complex (i.e., Cut Probe 1), region 1 islocated upstream of region 2 and region 2 is located upstream of region3. As for other embodiments of invasive cleavage, the region shown as“2” can represent a region where there is a physical, but not sequence,overlap between the INVADER portion of the Cut Probe 1 and the Probe 2oligonucleotide.

[0464] The cleavage products of the secondary invasive cleavage reaction(i.e., Cut Probe 2) can either be detected, or can in turn be designedto constitute yet another integrated INVADER-target complex to be usedwith a third probe molecule, again unrelated to the preceding targets.

[0465] The present invention is not limited to the configurationsdiagrammed in FIGS. 96 and 97. It is envisioned that the oligonucleotideproduct of a primary cleavage reaction may fill the role of any of theoligonucleotides described herein (e.g., it may serve as a target strandwithout an attached INVADER oligonucleotide-like sequence, or it mayserve as a stacker oligonucleotide, as described above), to enhance theturnover rate seen in the secondary reaction by stabilizing the probehybridization through coaxial stacking.

[0466] Secondary cleavage reactions in some preferred embodiments of thepresent invention include the use of FRET cassettes such as thosedescribed in Examples 54 through 62. Such molecules provide both asecondary target and a FRET labeled cleavable sequence, allowinghomogeneous detection (i.e., without product separation or othermanipulation after the reaction) of the sequential invasive cleavagereaction. Other preferred embodiments use a secondary reaction system inwhich the FRET probe and synthetic target are provided as separateoligonucleotides.

[0467] In a preferred embodiment, each subsequent reaction isfacilitated by (i.e., is dependent upon) the product of the previouscleavage, so that the presence of the ultimate product may serve as anindicator of the presence of the target analyte. However, cleavage inthe second reaction need not be dependent upon the presence of theproduct of the primary cleavage reaction; the product of the primarycleavage reaction may merely measurably enhance the rate of the secondcleavage reaction.

[0468] In summary, the INVADER assay cascade (i.e., sequential invasivecleavage reactions) of the present invention is a combination of two ormore linear assays that allows the accumulation of the ultimate productat an exponential rate, but without significant risk of carryovercontamination. It is an important to note that background that does notarise from sequential cleavage, such as thermal breakage of thesecondary probe, generally increases linearly with time. In contrast,signal generation from a 2-step sequential reaction follows quadratickinetics. Thus, collection of data as a time course, either by takingtime points or through the use of an instrument that allows real-timedetection during the INVADER assay reaction incubations, provides theattractive capability of discriminating between the true signal and anybackground solely on the basis of quadratic versus linear increases insignal over time. For example, when viewed graphically, the real signalwill appear as a quadratic curve, while any accumulating background willbe linear, and thus easy to distinguish, even if the absolute level ofthe background signal (e.g., fluorescence in a FRET detection format) issubstantial.

[0469] The sequential invasive cleavage amplification of the presentinvention can be used as an intermediate boost to any of the detectionmethods (e.g., gel based analysis by either standard or by chargereversal), polymerase tailing, and incorporation into a protein bindingregion, described herein. When used is such combinations the increasedproduction of a specific cleavage product in the invasive cleavage assayreduces the burdens of sensitivity and specificity on the read-outsystems, thus facilitating their use.

[0470] In addition to enabling a variety of detection platforms, thecascade strategy is suitable for multiplex analysis of individualanalytes (i.e., individual target nucleic acids) in a single reaction.The multiplex format can be categorized into two types. In one case, itis desirable to know the identity (and amount) of each of the analytesthat can be present in a clinical sample, or the identity of each of theanalytes as well as an internal control. To identify the presence ofmultiple individual analytes in a single sample, several distinctsecondary amplification systems may be included. Each probe cleaved inresponse to the presence of a particular target sequence (or internalcontrol) can be designed to trigger a different cascade coupled todifferent detectable moieties, such as different sequences to beextended by DNA polymerase or different dyes in an FRET format. Thecontribution of each specific target sequence to final product canthereby be tallied, allowing quantitative detection of different genesor alleles in a sample containing a mixture of genes or alleles.

[0471] In the second configuration, it is desirable to determine if anyof several analytes are present in a sample, but the exact identity ofeach does not need to be known. For example, in blood banking it isdesirable to know if any one of a host of infectious agents is presentin a sample of blood. Because the blood is discarded regardless of whichagent is present, different signals on the probes would not be requiredin such an application of the present invention, and may actually beundesirable for reasons of confidentiality. In this case, the 5′ arms(i.e., the 5′ portion which will be released upon cleavage) of thedifferent analyte-specific probes would be identical and would thereforetrigger the same secondary signal cascade. A similar configuration wouldpermit multiple probes complementary to a single gene to be used toboost the signal from that gene or to ensure inclusivity when there arenumerous alleles of a gene to be detected.

[0472] In the primary INVADER reaction, there are two potential sourcesof background. The first is from INVADER-independent cleavage of probeannealed to the target, to itself, or to one of the otheroligonucleotides present in the reaction. It can be seen byconsideration of FIGS. 96 and 97 that the probes of the primary cleavagereactions depicted are designed to have regions of complementarity tothe other oligonucleotides involved in the subsequent reactions, and, asdepicted in FIG. 97, to other regions of the same molecule. The use ofan enzyme that cannot efficiently cleave a structure that lacks a primer(e.g., that cannot cleave the structures diagrammed in FIG. 16A or 16D)is preferred for this reason. As shown in FIGS. 99 and 100, the enzymePfu FEN-1 gives no detectable cleavage in the absence of the upstreamoligonucleotide or even in the presence of an upstream oligonucleotidethat fails to invade the probe-target complex. This indicates that thePfu FEN-1 endonuclease is a suitable enzyme for use in the methods ofthe present invention.

[0473] Other structure-specific nucleases may be suitable as a well. Asdiscussed in the first example, some 5′ nucleases can be used inconditions that significantly reduce this primer-independent cleavage.For example, it has been shown that when the 5′ nuclease of DNAPTaq isused to cleave hairpins the primer-independent cleavage is markedlyreduced by the inclusion of a monovalent salt in the reaction(Lyamichev, et al., [1993], supra).

[0474] Test for INVADER Oligonucleotide-Independent Cleavage

[0475] A simple test can be performed for any enzyme in combination withany reaction buffer to gauge the amount of INVADERoligonucleotide-independent cleavage to be expected from thatcombination. A small hairpin-like test molecule that can be used with orwithout a primer hybridized to a 3′ arm, the S-60 molecule, is depictedin FIG. 30. The S-60 and the oligonucleotide P15 are a convenient set ofmolecules for testing the suitability of an enzyme for application inthe present invention and conditions for using these molecules aredescribed in Example 11. Other similar hairpins may be used. A cleavagestructure may be assembled from separate oligonucleotides as diagrammedin FIGS. 99a-e. Reactions using these structures to examine the activityof the Pfu FEN-1 enzyme in the presence or absence of an upstreamoverlapping oligonucleotide are described in Example 45 and the resultsare displayed in FIG. 100. To test any particular combination of enzymeand cleavage conditions, similar reactions can be assembled. Outside ofthe variables of reaction conditions to be tested for any particularenzyme (e.g., salt sensitivities, divalent cation requirements) the testreactions should accommodate any known limitations of the test enzyme.For example, the test reactions should be performed at a temperaturethat is within the operating temperature range of the candidate enzyme,if known.

[0476] It is not necessary that multiple lengths of overlap bedemonstrated for each candidate enzyme, but the activity of the enzymein the absence of an upstream oligonucleotide (sequence or physicaloverlap) (as shown in FIG. 99a) and in the presence of anoligonucleotide that does not overlap (FIG. 99b) should be assessed. Itis preferable that structures lacking an upstream oligonucleotide becleaved at less than one half of the rate seen in the presence of anupstream overlapping oligonucleotide. It is more preferable that thesestructures be cleaved at less than about on tenth the rate of theinvasive cleavage structure. It is most preferred that cleavage of thesestructures occur at less than one percent the rate of the invasivecleavage structure.

[0477] If the cleaved product is to serve as an upstream oligonucleotidein a subsequent cleavage reaction, as diagrammed in FIG. 96, the mostrapid reaction will be achieved if the other components of the secondcleavage structure (i.e., Target 2 and Probe 2 in FIG. 96) are providedin excess compared to the amount of first cleavage product, so thatcleavage may proceed immediately after the upstream oligonucleotide(i.e, Cut Probe 1 in FIG. 96) is made available. To provide an abundanceof the second target strand or cassette (Target 2 in FIG. 96) one mayuse an isolated natural nucleic acid, such as bacteriophage M13 DNA, orone may use a synthetic oligonucleotide. If a synthetic oligonucleotideis chosen as the second target sequence, the sequence employed should beexamined for regions of unintended self-complementarity (similarconsiderations apply to short isolated natural nucleic acids such asrestriction enzyme fragments or PCR products; natural nucleic acidtargets whose 3′ end is located ≧100 nucleotides downstream of the probebinding site on the target strand are generally long enough to obviatethe design considerations discussed below). Specifically, it should bedetermined that the 3′ end of the synthetic oligonucleotide may nothybridize to the target strand (i.e., intra-strand hybridization)upstream of the probe, triggering unintended cleavage. Simpleexamination of the sequence of the synthetic oligonucleotide shouldreveal if the 3′ end has sufficient complementarity to the region of thetarget upstream of the probe binding site to pose a problem (i.e, itwould reveal whether the synthetic oligonucleotide can form a hairpin atits 3′ end which could act as an invading oligonucleotide to causecleavage of the 2^(nd) probe in the absence of the hybridization of theintended INVADER oligonucleotide (i.e., the cleavage product from thefirst invasive cleavage reaction)). If 3 or more of the last 4 to 7nucleotides (the 3′ terminal region) of the synthetic target canbasepair upstream of the probe such that there is an overlap with theprobe-target duplex, or such that the duplexes formed by the synthetictarget strand with its own 3′ terminal region and with the probe abutwithout a gap and the 3′ terminal region has an additional 1 or 2nucleotides unpaired at the extreme 3′ end of the synthetic target, thenthe sequence of the synthetic target oligonucleotide should be modified.The sequence may be changed to disrupt the interaction of the 3′terminal region or to increase the distance between the probe bindingsite and the regions to which the 3′ terminus is binding. Alternatively,the 3′ end may be modified to reduce its ability to direct cleavage(e.g., by adding a 3′ phosphate during synthesis) (see Ex. 35, Table 3)or by adding several additional nucleotides that will not basepair in aself-complementary manner (i.e., they will not participate in theformation of a hairpin structure).

[0478] When the product of a first invasive cleavage reaction isdesigned to form a target that can fold on itself to direct cleavage ofa second probe, the IT complex as diagrammed in FIG. 97, the design ofthe sequence used to form the stem/loop of the IT complex should beconsidered. To be factored into the design of such a probe are 1) thelength of the region of self-complementarity, 2) the type of overlap(i.e., what 3′ moiety) and, if an overlap in sequence is selected, thelength of the region of overlap (region “X” in FIG. 25) and 3) thestability of the hairpin or stern/loop structure as predicted by bothWatson-Crick base pairing and by the presence or absence of aparticularly stable loop sequence (e.g., a tetraloop [Tinoco et al.,supra], or a triloop [Hirao et al., supra]). It is desirable that thissequence have nucleotides that can base pair (intrastrand), so that thesecond round of invasive cleavage may occur, but that the structure notbe so strong that its presence will prevent the cleavage of the probe inthe primary reaction (i.e., Probe 1 in FIG. 96). As shown herein, thepresence of a secondary structure in the 5′ arm of a cleavage structurecleaved by a structure-specific nuclease may inhibit cleavage by somestructure-specific nucleases (Ex. 1).

[0479] The length of the region of self-complementarity within Probe 1determines the length of the region of the duplex upstream of Probe 2 inthe second cleavage structure (see FIG. 97). Different enzymes havedifferent length requirements for this duplex to effect invasivecleavage efficiently. For example, the Pfu FEN-1 and Mja FEN-1 enzymeshave been tested for the effect of this duplex length using the set oftarget/INVADER oligonucleotide molecules depicted in FIG. 98 (i.e., SEQID NOS: 118, 119, 147-151). The invasive cleavage reactions wereperformed as described in Example 38, using 1 pM IT3 (SEQ ID NO: 118), 2μM probe PR1 (SEQ ID NO: 119) for 5 min, and the rates of cleavage areshown in Table 2. TABLE 2 Length of Pfu FEN-1 Turnover, Mja FEN-1Turnover, Duplex per min. per min. 0 0 0 3 1 29 4 10 57 6 44 51 8 45 46

[0480] The data shown in Table 2 demonstrate that the Pfu FEN-1 enzymecan be used with stems of 3 or 4 bases, but that the rate of cleavage ismaximized when the stem is greater than 4 basepairs in length. Table 2shows that the Mja FEN-1 enzyme can cleave efficiently using shorterstems; however, as this enzyme can also cleave a probe in the absence ofan upstream oligonucleotide, Mja FEN-1 is not preferred for use in thesequential invasive cleavage methods of the present invention.

[0481] A similar test can be performed using any candidate enzyme todetermine how much self-complementarity may be designed into theProbe 1. The use of a shorter stem means that the overall probe may beshorter. This is beneficial because shorter probes are less costly tosynthesize, and because shorter probes will have fewer sequences thatmight form unintended intrastrand structures. In assessing the activityof a candidate enzyme on the structures such as those shown in FIG. 98it is not required that the stem length chosen allow the maximum rate ofcleavage to occur. For example, in considering the case of Pfu FEN-1,the advantages of using a 4 basepair stem (e.g., cost or sequencelimitations), with a cleavage rate of 10 cleavages per minute, mayoutweigh the rate advantage of using a longer 6 basepair stem (44cleavages/min.), in the context of a particular experiment. It is withinthe scope of the present invention that some elements chosen for use inthe assay be sub-optimal for performance of that particular element, ifthe use of a sub-optimal design benefits the objectives of thatparticular experiment as a whole.

[0482] In designing oligonucleotides to be employed as a probe that,once cleaved, forms a stem-loop structure as diagrammed in FIG. 97(i.e., Probe 1 in FIG. 97), it has been found that the stability of theloop is not a factor in the efficiency of cleavage of either Probe 1 orProbe 2. Loops tested have included stable triloops, loops of 3 and 4nucleotides that were not predicted to be particularly stable (i.e., thestability is determined by the duplex sequence and not by additionalstabilizing interactions within the loop), and large loops of up toabout 25 nucleotides.

[0483] IV. Fractionation of Specific Nucleic Acids by Selective ChargeReversal

[0484] Some nucleic acid-based detection assays involve the elongationand/or shortening of oligonucleotide probes. For example, as describedherein, the primer-directed, primer-independent, and INVADER-directedcleavage assays, as well as the “nibbling” assay all involve thecleavage (i.e., shortening) of oligonucleotides as a means for detectingthe presence of a target nucleic sequence. Examples of other detectionassays that involve the shortening of an oligonucleotide probe includethe “TaqMan” or nick-translation PCR assay described in U.S. Pat. No.5,210,015 to Gelfand et al. (the disclosure of which is hereinincorporated by reference), the assays described in U.S. Pat. Nos.4,775,619 and 5,118,605 to Urdea (the disclosures of which are hereinincorporated by reference), the catalytic hybridization amplificationassay described in U.S. Pat. No. 5,403,711 to Walder and Walder (thedisclosure of which is herein incorporated by reference), and thecycling probe assay described in U.S. Pat. Nos. 4,876,187 and 5,011,769to Duck et al. (the disclosures of which are herein incorporated byreference). Examples of detection assays that involve the elongation ofan oligonucleotide probe (or primer) include the polymerase chainreaction (PCR) described in U.S. Pat. Nos. 4,683,195 and 4,683,202 toMullis and Mullis et al. (the disclosures of which are hereinincorporated by reference) and the ligase chain reaction (LCR) describedin U.S. Pat. Nos. 5,427,930 and 5,494,810 to Birkenmeyer et al. andBarany et al. (the disclosures of which are herein incorporated byreference). The above examples are intended to be illustrative ofnucleic acid-based detection assays that involve the elongation and/orshortening of oligonucleotide probes and do not provide an exhaustivelist.

[0485] Typically, nucleic acid-based detection assays that involve theelongation and/or shortening of oligonucleotide probes requirepost-reaction analysis to detect the products of the reaction. It iscommon that the specific reaction product(s) must be separated from theother reaction components, including the input or unreactedoligonucleotide probe. One detection technique involves theelectrophoretic separation of the reacted and unreacted oligonucleotideprobe. When the assay involves the cleavage or shortening of the probe,the unreacted product will be longer than the reacted or cleavedproduct. When the assay involves the elongation of the probe (orprimer), the reaction products will be greater in length than the input.Gel-based electrophoresis of a sample containing nucleic acid moleculesof different lengths separates these fragments primarily on the basis ofsize. This is due to the fact that in solutions having a neutral oralkaline pH, nucleic acids having widely different sizes (i.e.,molecular weights) possess very similar charge-to-mass ratios and do notseparate (Andrews, Electrophoresis, 2nd Edition, Oxford University Press(1986), pp. 153-154]. The gel matrix acts as a molecular sieve andallows nucleic acids to be separated on the basis of size and shape(e.g., linear, relaxed circular or covalently closed supercoiledcircles).

[0486] Unmodified nucleic acids have a net negative charge due to thepresence of negatively charged phosphate groups contained within thesugar-phosphate backbone of the nucleic acid. Typically, the sample isapplied to gel near the negative pole and the nucleic acid fragmentsmigrate into the gel toward the positive pole with the smallestfragments moving fastest through the gel.

[0487] The present invention provides a novel means for fractionatingnucleic acid fragments on the basis of charge. This novel separationtechnique is related to the observation that positively charged adductscan affect the electrophoretic behavior of small oligonucleotidesbecause the charge of the adduct is significant relative to charge ofthe whole complex. In addition to the use of positively charged adducts(e.g., Cy3 and Cy5 fluorescent dyes, the positively chargedheterodimeric DNA-binding dyes shown in FIG. 66, etc.), theoligonucleotide may contain amino acids (particularly useful amino acidsare the charged amino acids: lysine, arginine, asparate, glutamate),modified bases, such as amino-modified bases, and/or a phosphonatebackbone (at all or a subset of the positions). In other embodiments, asdiscussed further below, a neutral dye or detection moiety (e.g.,biotin, streptavidin, etc.) may be employed in place of a positivelycharged adduct, in conjunction with the use of amino-modified basesand/or a complete or partial phosphonate backbone.

[0488] This observed effect is of particular utility in assays based onthe cleavage of DNA molecules. Using the assays described herein as anexample, when an oligonucleotide is shortened through the action of aCLEAVASE enzyme or other cleavage agent, the positive charge can be madeto not only significantly reduce the net negative charge, but toactually override it, effectively “flipping” the net charge of thelabeled entity. This reversal of charge allows the products oftarget-specific cleavage to be partitioned from uncleaved probe byextremely simple means. For example, the products of cleavage can bemade to migrate towards a negative electrode placed at any point in areaction vessel, for focused detection without gel-basedelectrophoresis; Example 24 provides examples of devices suitable forfocused detection without gel-based electrophoresis. When a slab gel isused, sample wells can be positioned in the center of the gel, so thatthe cleaved and uncleaved probes can be observed to migrate in oppositedirections. Alternatively, a traditional vertical gel can be used, butwith the electrodes reversed relative to usual DNA gels (i.e., thepositive electrode at the top and the negative electrode at the bottom)so that the cleaved molecules enter the gel, while the uncleaveddisperse into the upper reservoir of electrophoresis buffer.

[0489] An important benefit of this type of readout is the absolutenature of the partition of products from substrates (i.e., theseparation is virtually 100%). This means that an abundance of uncleavedprobe can be supplied to drive the hybridization step of the probe-basedassay, yet the unconsumed (i.e., unreacted) probe can, in essence, besubtracted from the result to reduce background by virtue of the factthat the unreacted probe will not migrate to the same pole as thespecific reaction product.

[0490] Through the use of multiple positively charged adducts, syntheticmolecules can be constructed with sufficient modification that thenormally negatively charged strand is made nearly neutral. When soconstructed, the presence or absence of a single phosphate group canmean the difference between a net negative or a net positive charge.This observation has particular utility when one objective is todiscriminate between enzymatically generated fragments of DNA, whichlack a 3′ phosphate, and the products of thermal degradation, whichgenerally retain a 3′ phosphate (and thus two additional negativecharges). Examples 22 and 23 demonstrate the ability to separatepositively charged reaction products from a net negatively chargedsubstrate oligonucleotide. As discussed in these examples,oligonucleotides may be transformed from net negative to net positivelycharged compounds. In Example 23, the positively charged dye, Cy3 wasincorporated at the 5′ end of a 22-mer (SEQ ID NO: 50) which alsocontained two amino-substituted residues at the 5′ end of theoligonucleotide; this oligonucleotide probe carries a net negativecharge. After cleavage, which occurred 2 nucleotides into the probe, thefollowing labeled oligonucleotide was released: 5′-Cy3-AminoT-AminoT-3′(in addition to unlabeled fragment comprising the remaining 20nucleotides of SEQ ID NO: 50). This short fragment bears a net positivecharge while the remainder of the cleaved oligonucleotide and theunreacted or input oligonucleotide bear net negative charges.

[0491] The present invention contemplates embodiments wherein thespecific reaction product produced by any cleavage of anyoligonucleotide can be designed to carry a net positive charge while theunreacted probe is charge neutral or carries a net negative charge. Thepresent invention also contemplates embodiments where the releasedproduct may be designed to carry a net negative charge while the inputnucleic acid carries a net positive charge. Depending on the length ofthe released product to be detected, positively charged dyes may beincorporated at the one end of the probe and modified bases may beplaced along the oligonucleotide such that upon cleavage, the releasedfragment containing the positively charged dye carries a net positivecharge. Amino-modified bases may be used to balance the charge of thereleased fragment in cases where the presence of the positively chargedadduct (e.g., dye) alone is not sufficient to impart a net positivecharge on the released fragment. In addition, the phosphate backbone maybe replaced with a phosphonate backbone at a level sufficient to imparta net positive charge (this is particularly useful when the sequence ofthe oligonucleotide is not amenable to the use of amino-substitutedbases); FIGS. 45 and 46 show the structure of short oligonucleotidescontaining a phosphonate group on the second T residue). Anoligonucleotide containing a fully phosphonate-substituted backbonewould be charge neutral (absent the presence of modified chargedresidues bearing a charge or the presence of a charged adduct) due tothe absence of the negatively charged phosphate groups.Phosphonate-containing nucleotides (e.g., methylphosphonate-containingnucleotides are readily available and can be incorporated at anyposition of an oligonucleotide during synthesis using techniques whichare well known in the art.

[0492] In essence, the invention contemplates the use of charge-basedseparation to permit the separation of specific reaction products fromthe input oligonucleotides in nucleic acid-based detection assays. Thefoundation of this novel separation technique is the design and use ofoligonucleotide probes (typically termed “primers” in the case of PCR)which are “charge balanced” so that upon either cleavage or elongationof the probe it becomes “charge unbalanced,” and the specific reactionproducts may be separated from the input reactants on the basis of thenet charge.

[0493] In the context of assays that involve the elongation of anoligonucleotide probe (i.e., a primer), such as is the case in PCR, theinput primers are designed to carry a net positive charge. Elongation ofthe short oligonucleotide primer during polymerization will generate PCRproducts that now carry a net negative charge. The specific reactionproducts may then easily be separated and concentrated away from theinput primers using the charge-based separation technique describedherein (the electrodes will be reversed relative to the description inExample 23 as the product to be separated and concentrated after a PCRwill carry a negative charge).

[0494] V. Signal Enhancement by Tailing of Reaction Products in theINVADER Oligonucleotide-Directed Cleavage Assay

[0495] It has been determined that when oligonucleotide probes are usedin cleavage detection assays at elevated temperature, some fraction ofthe truncated probes will have been shortened by nonspecific thermaldegradation, and that such breakage products can make the analysis ofthe target-specific cleavage data more difficult. The thermaldegradation that creates a background ladder of bands when the probes ofthe present invention are treated at high temperature for more than afew minutes occurs as a two step process. In the first step theN-glycosyl bond breaks, leaving an abasic site in the DNA strand. At theabasic site the DNA chain is weakened and undergoes spontaneous cleavagethrough a beta-elimination process. It has been determined that purinebases are about 20 times more prone to breakage than pyrimidine bases(Lindahl, Nature 362:709 [1993]). This suggests that one way of reducingbackground in methods using oligonucleotides at elevated temperatures isto select target sequences that allow the use of pyrimidine-rich probes.It is preferable, where possible, to use oligonucleotides that areentirely composed of pyrimidine residues. If only one or a few purinesare used, the background breakage will appear primarily at thecorresponding sites, and these bands (due to thermal breakdown) may bemistaken for the intended cleavage products if care is not taken in thedata analysis (i.e., proper controls must be run).

[0496] Background cleavage due to thermal breakdown of probeoligonucleotides can, when not resolved from specific cleavage products,reduce the accuracy of quantitation of target nucleic acids based on theamount of accumulated product in a set timeframe. One means ofdistinguishing the specific from the nonspecific products is disclosedabove, and is based on partitioning the products of these reactions bydifferences in the net charges carried by the different molecularspecies in the reaction. As was noted in that discussion, the thermalbreakage products usually retain 3′ phosphates after breakage, while theenzyme-cleaved products do not. The two negative charges on thephosphate facilitate charge-based partition of the products.

[0497] The absence of a 3′ phosphate on the desired subset of the probefragments may be used to advantage in enzymatic assays as well. Nucleicacid polymerases, both non-templated (e.g., terminal deoxynucleotidyltransferase, polyA polymerase) and template-dependent (e.g., Pol I-typeDNA polymerases), require an available 3′ hydroxyl by which to attachfurther nucleotides. This enzymatic selection of 3′ end structure may beused as an effective means of partitioning specific from non-specificproducts.

[0498] In addition to the benefits of the partitioning described above,the addition of nucleotides to the end of the specific product of anINVADER oligonucleotide-specific cleavage offers an opportunity toeither add label to the products, to add capturable tails to facilitatesolid-support based readout systems, or to do both of these things atthe same time. Some possible embodiments of this concept are illustratedin FIG. 56.

[0499] In FIG. 56, an INVADER cleavage structure comprising an INVADERoligonucleotide containing a blocked or non-extendible 3′ end (e.g., a3′ dideoxynucleotide) and a probe oligonucleotide containing a blockedor non-extendable 3′ end (the open circle at the 3′ end of theoligonucleotides represents a non-extendible nucleotide) and a targetnucleic acid is shown; the probe oligonucleotide may contain a 5′ endlabel such as a biotin or a fluorescein (indicated by the stars) label(cleavage structures which employ a 5′ biotin-labeled probe or a 5′fluorescein-labeled probe are shown below the large diagram of thecleavage structure to the left and the right, respectively). Followingcleavage of the probe (the site of cleavage is indicated by the largearrowhead), the cleaved biotin-labeled probe is extended using atemplate-independent polymerase (e.g., TdT) and fluoresceinatednucleotide triphosphates. The fluorescein tailed cleaved probe moleculeis then captured by binding via its 5′ biotin label to streptavidin andthe fluorescence is then measured. Alternatively, following, cleavage ofa 5′-fluoresceinated probe, the cleaved probe is extended using atemplate-independent polymerase (e.g., TdT) and dATP. The polyadenylated(A-tailed) cleaved probe molecule is then captured by binding via thepolyA tail to oligo dT attached to a solid support.

[0500] The examples described in FIG. 56 are based on the use of TdT totail the specific products of INVADER-directed cleavage. The descriptionof the use of this particular enzyme is presented by way of example andis not intended as a limitation (indeed, when probe oligonucleotidescomprising RNA are employed, cleaved RNA probes may be extended usingpolyA polymerase). It is contemplated that an assay of this type can beconfigured to use a template-dependent polymerase, as described above.While this would require the presence of a suitable copy templatedistinct from the target nucleic acid, on which the truncatedoligonucleotide could prime synthesis, it can be envisaged that a probethat before cleavage would be unextendible, due to either mismatch ormodification of the 3′ end, could be activated as a primer when cleavedby an INVADER oligonucleotide-directed cleavage. A template directedtailing reaction also has the advantage of allowing greater selectionand control of the nucleotides incorporated.

[0501] The use of nontemplated tailing does not require the presence ofany additional nucleic acids in the detection reaction, avoiding onestep of assay development and troubleshooting. In addition, the use ofnon-templated synthesis eliminated the step of hybridization,potentially speeding up the assay. Furthermore, the TdT enzyme is fast,able to add at least >700 nucleotides to substrate oligonucleotides in a15 minute reaction.

[0502] As mentioned above, the tails added can be used in a number ofways. It can be used as a straight-forward way of adding labeledmoieties to the cleavage product to increase signal from each cleavageevent. Such a reaction is depicted in the left side of FIG. 66. Thelabeled moieties may be anything that can, when attached to anucleotide, be added by the tailing enzyme, such as dye molecules,haptens such as digoxigenin, or other binding groups such as biotin.

[0503] In a preferred embodiment the assay includes a means ofspecifically capturing or partitioning the tailed INVADERoligonucleotide-directed cleavage products in the mixture. It can beseen that target nucleic acids in the mixture may be tailed during thereaction. If a label is added, it is desirable to partition the tailedINVADER oligonucleotide-directed cleavage products from these otherlabeled molecules to avoid background in the results. This is easilydone if only the cleavage product is capable of being captured. Forexample, consider a cleavage assay of the present invention in which theprobe used has a biotin on the 5′ end and is blocked from extension onthe 3′ end, and in which a dye is added during tailing. Consider furtherthat the products are to be captured onto a support via the biotinmoiety, and the captured dye measured to assess the presence of thetarget nucleic acid. When the label is added by tailing, only thespecifically cleaved probes will be labeled. The residual uncut probescan still bind in the final capture step, but they will not contributeto the signal. In the same reaction, nicks and cuts in the targetnucleic acid may be tailed by the enzyme, and thus become dye labeled.In the final capture these labeled targets will not bind to the supportand thus, although labeled, they will not contribute to the signal. Ifthe final specific product is considered to consist of two portions, theprobe-derived portion and the tail portion, it can be seen from thisdiscussion that it is particularly preferred that, when theprobe-derived portion is used for specific capture, whether byhybridization, biotin/streptavidin, or other method, that the label beassociated with the tail portion. Conversely, if a label is attached tothe probe-derived portion, then the tail portion may be made suitablefor capture, as depicted on the right side of FIG. 66. Tails may becaptured in a number of ways, including hybridization, biotinincorporation with streptavidin capture, or by virtue if the fact thatthe longer molecules bind more predictably and efficiently to a numberof nucleic acid minding matrices, such as nitrocellulose, nylon, orglass, in membrane, paper, resin, or other form. While not required forthis assay, this separation of functions allows effective exclusion fromsignal of both unreacted probe and tailed target nucleic acid.

[0504] In addition to the supports described above, the tailed productsmay be captured onto any support that contains a suitable capturemoiety. For example, biotinylated products are generally captured withavidin-treated surfaces. These avidin surfaces may be in microtitreplate wells, on beads, on dipsticks, to name just a few of thepossibilities. Such surfaces can also be modified to contain specificoligonucleotides, allowing capture of product by hybridization. Capturesurfaces as described herein are generally known to those skilled in theart and include nitrocellulose dipsticks (e.g., GENECOMB, BioRad,Hercules, Calif.).

[0505] VI. Signal Enhancement by Completion of an Activated ProteinBinding Site

[0506] In addition to the DNA polymerase tailing reaction describedabove, the present invention also contemplates the use of the productsof the invasive cleavage reaction to form activated protein bindingsites, such as RNA polymerase promoter duplexes, thereby allowing theinteraction of the completed site to be used as an indicator of thepresence of the nucleic acid that is the target of the invasive cleavagereaction. By way of example, when an RNA polymerase promoter duplex isactivated by being made complete (i.e., double-stranded over thatportion of the promoter region required for polymerase binding) throughthe hybridization of the oligonucleotide product of the invasivecleavage reaction, the synthesis of RNA can be used as such anindicator.

[0507] It is not intended that the transcription reaction of the presentinvention be limited to the use of any particular RNA polymerase or RNApolymerase promoter region. Promoter sequences are well characterizedfor several bacteriophage, including bacteriophage SP6, T7 and T3. Inaddition, promoter sequences have been well characterized for a numberof both eukaryotic and prokaryotic RNA polymerases. In a preferredembodiment, the promoter used enables transcription from one of thebacteriophage RNA polymerases. In a particularly preferred embodiment,the promoter used enables transcription by T7 RNA polymerase. Means ofperforming transcription in vitro are well known in the art andcommercial kits are available for performing transcription witheukaryotic, prokaryotic or bacteriophage RNA polymerases (e.g., fromPromega Corp., Madison, Wis.).

[0508] The protein binding regions of the present invention are notlimited to the bacteriophage RNA polymerase promoters described above.Other promoter sequences that are contemplated are those of prokaryotesand eukaryotes. For example, many strains of bacteria and fungi are usedfor the expression of heterologous proteins. The minimal promotersrequired for transcription by the RNA polymerases of organisms such asyeast and other fungi, eubacteria, nematodes, and cultured mammaliancells are well described in the literature and in the catalogs ofcommercial suppliers of DNA vectors for the expression of foreignproteins in these organisms.

[0509] The binding sites for other types of nucleic acid (e.g., DNA)binding proteins are contemplated for use in the present invention. Forexample, proteins involved in the regulation of genes exert theireffects by binding to the DNA in the vicinity of the promoter from whichthe RNA from that gene is transcribed. The lac operator of E. coli isone example of a particularly well characterized and commonly used generegulation system in which the lac repressor protein binds to specificsequences that overlap, and thus block, the promoter for the genes underthe repressor's control (Jacob and Monod, Cold Spring Harbor Symposiumon Quantitative Biol. XXVI:193-211 [1961]). Many similar systems havebeen described in bacteria, including the trp and AraC regulatorysystems. Given the large amount of information available about bacterialpromoters, the steps described below for the design of suitable partialpromoters for the bacteriophage RNA polymerases can be readily adaptedto the design of detection systems based on these other promoters.

[0510] As noted above, many of the bacterial promoters are under thecontrol of a repressor or other regulatory protein. It is considered tobe within the scope of the present invention to include the creation ofcomposite binding sites for these regulatory proteins through theprovision of a nucleic acid fragment (e.g., a non-target cleavageproduct generated in an invasive cleavage reaction). The binding of theregulatory protein to the completed protein binding region (e.g., thecomposite binding region) can be assessed by any one of a number ofmeans, including slowed electrophoretic migration of either the proteinor the DNA fragment, or by a conformational change in the protein or DNAupon binding. In addition, transcription from a downstream promoter canbe monitored for up- or down-regulation as a result of the binding ofthe regulatory protein to the completed protein binding region.

[0511] In addition to the bacterial systems described above, many genesin eukaryotic systems have also been found to be under the control ofspecific proteins that bind to specific regions of duplex DNA. Examplesinclude, but are not limited to, the OCT-1, OCT-2 and AP-4 proteins inmammals and the GAL4 and GCN4 proteins in yeast. Such regulatoryproteins usually have a structural motif associated with duplex nucleicacid binding, such as a helix-turn-helix, a zinc finger or a leucinezipper [for review, see, Molecular and Cellular Biology, Wolfe (Ed.),Wadsworth Publishing Co., Belmont, Calif., pp. 694-715 [1993]).

[0512] For simplicity the test reaction described here will refer to T7RNA polymerase, and its promoter. This is not intended to limit theinvention to the use of this RNA polymerase, and those skilled in theart of molecular biology would be able to readily adapt this describedtest to the examination of any of the DNA binding proteins, RNApolymerases and their binding or promoter sites discussed above.

[0513] It is known in the art that active T7 promoters can be formed bythe hybridization of two oligonucleotides, each comprising either thetop or bottom strand of the promoter sequence, such that a completeun-nicked duplex promoter is formed (Milligan et al., Nucl. Acids Res.,15:21, 8783-8798 (1987)]. The present invention shows that one way ofmaking the initiation of transcription dependent on the products of aninvasive cleavage reaction is to design the probe for the cleavagereaction such that a portion of an RNA polymerase promoter is releasedas product. The remaining DNA piece or pieces required to assemble apromoter duplex may either be provided as elements in the reactionmixture, or they may be produced by other invasive cleavage events. Ifthe oligonucleotide pieces are designed to comprise appropriate regionsof complementarity they may base pair to form a complete promoter duplexcomposed of three or more nucleic acid fragments, as depicted in FIG.88B. A promoter assembled in this way will have nicks in the backbone ofone or both strands. In one embodiment, these nicks may be covalentlyclosed through the use of a DNA ligase enzyme. In a preferredembodiment, the nicks are positioned such that transcription can proceedwithout ligation. In selecting the site of a nick created by theassembly of the partial promoter fragment, at least one nick should bewithin the recognized promoter region for the RNA polymerase to be used.When a bacteriophage promoter is used, a nick should be betweennucleotides −17 and −1, measured from the site of transcriptioninitiation at +1. In a preferred embodiment, a nick will be betweennucleotides −13 and −8. In a particularly preferred embodiment, a nickwill be between nucleotides −12 and −10 on the non-template strand ofthe bacteriophage promoter.

[0514] When nicks are to be left unrepaired (i.e., not covalently closedwith a DNA ligase) it is important to assess the effect of the nicklocation on the level of transcription from the assembled promoter. Asimple test is to combine the oligonucleotides that comprise theseparate portions of the promoter with an oligonucleotide that comprisesone entire strand of the promoter to be assembled, thereby forming aduplex promoter with a nick in one strand. If the nick is in the top, ornon-template strand of the promoter, then the oligonucleotide thatcomprises the complete promoter is made to include additionalnon-promoter sequence on its 5′ end to serve as a template to be copiedin the transcription. This arrangement is depicted in FIG. 88B.Alternatively, if the nick is to be in the bottom, or template strand ofthe promoter, then the partial promoter oligonucleotide that covers the+1 position, the transcription start site, will include the additionaltemplate sequence. This arrangement is depicted in FIGS. 95A-D (thisFigure shows several different embodiments in which a cut probe ornon-target cleavage product is used to form a composite promoter whichcontains one or more nicks on the template strand). In either case, theseparate oligonucleotides are combined to form the complete promoter,and the assembly is used in a transcription reaction to create RNA.

[0515] To measure the effect of the nick, a substantially identicalpromoter fragment is created by hybridization of two oligonucleotidesthat each comprise one strand of the full-length promoter to create anun-nicked version of the same promoter. These two molecular assembliesare tested in parallel transcription reactions and the amount of theexpected RNA that is produced in each reaction is measured for both sizeand yield. A preferred method of assessing the size of the RNA is byelectrophoresis with subsequent visualization. If a labeled nucleotide(e.g., ³²P-GTP, or fluorescein-UTP) is used in the transcription, theRNA can be detected and quantitated by autoradiography, fluorescenceimaging or by transfer to support membrane with subsequent detection(e.g., by antibody or hybridization probing). Alternatively, ifunlabeled RNA is produced the amounts may be determined by other methodsknown in the art, such as by spectrophotometry or by electrophoresiswith subsequent staining and comparison to known standards.

[0516] If the size of the RNA is as predicted by the template sequence,or if it matches that produced from the control promoter, it can bepresumed to have initiated transcription at the same site in thecomplex, and to have produced essentially the same RNA product. If theproduct is much shorter then transcription is either initiating at aninternal site or is terminating prematurely (Schenborn and Mierendorf,Nucl. Acids Res., 13:17, 6223 [1985]; and Milligan et al., supra.).While this does not indicate that the assembly tested is completelyunsuitable for the assay, the partial transcripts will reduce the grossamount of RNA created, perhaps compromising the signal from the assay,and such products would require further characterization (e.g., fingerprinting or sequencing) to identify the nucleotide content of theproduct. It is preferred that the size of the RNA produced matches thatof the RNA produced in the control reaction.

[0517] The yield of the reaction is also examined. It is not necessarythat the level of transcription matches that of the control reaction. Insome instances (see Ex. 41, below) the nicked promoter may have anenhanced rate of transcription, while in other arrangementstranscription may be reduced (relative to the rate from the un-nickedpromoter assembly). It is only required that the amount of product bewithin the detection limits of the method to be used with the testpromoter.

[0518] It is reported that transcription from a bacteriophage promotercan produce 200 to 1000 copies of each transcription template (templateplus active promoter) in a reaction. These levels of transcription arenot required by the present invention. Reactions in which one RNA isproduced for each template are also contemplated.

[0519] The test described above will allow a promoter with a nick in anyposition to be assessed for utility in this assay. It is an objective ofthis invention to provide one or more of the oligonucleotides thatcomprise a partial promoter region through invasive cleavage event(s).In this embodiment, the partial promoter sequences are attached to theprobe oligonucleotide in the invasive cleavage assay, and are releasedby cleavage at specific site, as directed by the INVADERoligonucleotide. It is also intended that transcription be very poor ornonexistent in the absence of the correctly cleaved probe. To assess thesuccess of any oligonucleotide design at meeting these objectives,several transcription reaction tests can be performed.

[0520] For a promoter assembly that will have a nick on the non-templatestrand, several partial assemblies that should be tested are shown inFIGS. 86A-D. By way of example, but not by way of limitation, thisFigure depicts the tests for a nicked promoter in which the upstream, or5′ portion of the non-template strand is to be provided by the invasivecleavage assay. This fragment is seen in FIG. 86A labeled as “cutprobe”. Transcription reactions incubated in the presence of the duplexshown in FIG. 86A will test the ability of the upstream partial promoterto allow initiation of transcription when hybridized to a bottom strand,termed a “copy template.” Similarly, a reaction performed in thepresence of the duplex depicted in FIG. 86B will test the ability of thepartial promoter fragment nearest the initiation site (the +1 site, asindicated in FIG. 85B) to support transcription of the copy template. Itis an important feature of the present invention that neither of thesepartial promoter duplexes be able to support transcription at the samelevel as would by seen in transcription from an intact promoter asdepicted in FIG. 85B. It is preferred that neither of these partialpromoters be sufficient to initiate detectable transcription in the timecourse of an average transcription reaction (i.e., within about an hourof incubation).

[0521]FIGS. 86C and 86D depict two other duplex arrangements designed totest the effect of uncut probe within the transcription reaction. FIG.86C depicts the duplex formed between only the uncut probe and the copytemplate, while FIG. 86D includes the other portion of the promoter. The3′ region of the probe is not complementary to the promoter sequence andtherefore produces an unpaired branch in the middle of the promoter. Itis an important feature of the present invention that neither of thesebranched promoter duplexes be able to support transcription at the samelevel as would by seen in transcription from an intact promoter asdepicted in FIG. 85B. It is preferred that neither of these branchedpromoters be sufficient to initiate detectable transcription in the timecourse of an average transcription reaction (i.e., within about an hourof incubation).

[0522] In one embodiment of the transcription system of the presentinvention, the initiation of transcription from the copy template in theabsence of a complete promoter, or in the presence of a branchedpromoter, is prevented by the judicious placement of the nick or nicksin the composite promoter. For example, as shown in the examples below,placement of a nick between the −12 and −11 nucleotides of thenon-template strand of the bacteriophage T7 promoter allowstranscription to take place only when the probe has been successfullycut, as in an invasive cleavage reaction. However, in some instanceswhere the invasive cleavage reaction is to provide the upstream portionof the non-template strand of the promoter (e.g., as depicted in FIG.88B) it may be necessary or desirable to place the nick on that strandin a particular position for reasons other than providing an optimalcomposite promoter (i.e., one that is inactive in the absence of any oneof the promoter pieces). It may be necessary or desirable to place thenick in such a way that the creation of a branched complete promoter(FIG. 86D) has an undesirable level of transcription, reducingdependence of RNA production on the success of the invasive cleavagestep. It is shown in the examples below that transcription from such abranched promoter can be suppressed by a modification of the downstreamnon-template promoter piece, shown as the “Partial PromoterOligonucleotide” in FIGS. 86, 88, 90 and 95D. As depicted in FIG. 90,the partial promoter oligonucleotide can be provided with a 5′“tail” ofnucleotides that are not complementary to the template strand of thepromoter, but that are complementary to the 3′ portion of the probeoligonucleotide that would be removed in the invasive cleavage reaction.When uncut probe hybridizes to the copy template with the bound 5′tailed partial promoter oligonucleotide, the 5′ tail can basepair to the3′ region of the probe, forming a three-way junction as depicted in FIG.90A. This can effectively shut off transcription, as shown below. When acut probe hybridizes, as shown in FIG. 90B, a promoter with a smallbranch is formed, and it is shown herein that such a branched promotercan initiate transcription. Furthermore, if care is taken in selectingthe sequence of the 5′ tail (i.e., if the first unpaired base is thesame nucleotide at the 3′ nucleotide of the cut probe, so that theycompete for hybridization to the same template strand base), theresulting branched structure may also be cleaved by one of the structurespecific nucleases of the present invention, creating the un-branchedpromoter depicted in FIG. 90C, in some instances enhancing transcriptionover that seen with the FIG. 90B promoter.

[0523] The promoter duplex that is intended to be created, in thisembodiment, by the successful execution of the INVADER directed cleavageassay will include both the “cut probe” and the partial promoteroligonucleotide depicted in FIGS. 86A and B, aligned on a single copytemplate nucleic acid. The testing of the efficiency of transcription ofsuch a nicked promoter segment in comparison to the intact promoter isdescribed above. All of the oligonucleotides described for these testmolecules may be created using standard synthesis chemistries.

[0524] The set of test molecules depicted in FIG. 86 is designed toassess the transcription capabilities of the variety of structures thatmay be present in reactions in which the 5′ portion of the non-templatestrand of the promoter is to be supplied by the INVADER directedcleavage. It is also envisioned that a different portion of partialpromoter may be supplied by the invasive cleavage reaction (e.g., thedownstream segment of the non-template strand of the promoter), as isshown in FIG. 94. Portions of the template strand of the promoter mayalso be provided by the cut probe, as shown in FIGS. 95A-D. An analogousset of test molecules, including “cut” and uncut versions of the probeto be used in the invasive cleavage assay may be created to test anyalternative design, whether the nick is to be located on the template ornon template strand of the promoter.

[0525] The transcription-based visualization methods of the presentinvention may also be used in a multiplex fashion. Reactions can beconstructed such that the presence of one particular target leads totranscription from one type of promoter, while the presence of adifferent target sequence (e.g., a mutant or variant) or another targetsuspected of being present, may lead to transcription from a different(i.e., a second) type of promoter. In such an embodiment, the identityof the promoter from which transcription was initiated could be deducedfrom the type or size of the RNA produced.

[0526] By way of example, but not by way of limitation, thebacteriophage promoters can be compared with such an application inview. The promoters for the phage T7, T3 and SP6 are quite similar, eachbeing about 15 to 20 basepairs long, and sharing about 45% identitybetween −17 and −1 nucleotides, relative to the start of transcription.Despite these similarities, the RNA polymerases from these phage arehighly specific for their cognate promoters, such that the otherpromoters may be present in a reaction, but will not be transcribed(Chamberlin and Ryan, Enzymes XV:87-108 [1982]). Because these promotersare similar in size and in the way in which they are recognized by theirpolymerases (Li et al., Biochem. 35:3722 [1996]) similar nicked versionsof the promoters may be designed for use in the methods of the presentinvention by analogy to the examples described herein which employ theT7 promoter. Because of the high degree of specificity of the RNApolymerases, these nicked promoters may be used together to detectmultiple targets in a single reaction. There are many instances in whichit would be highly desirable to detect multiple nucleic acid targets ina single sample, including cases in which multiple infectious agents maybe present, or in which variants of a single type of target may need tobe identified. Alternatively, it is often desirable to use a combinationof probes to detect both a target sequence and an internal controlsequence, to gauge the effects of sample contaminants on the output ofthe assay. The use of multiple promoters allows the reaction to beassessed for both the efficiency of the invasive cleavage and therobustness of the transcription.

[0527] As stated above, the phage promoters were described in detail asan example of suitable protein binding regions (e.g., which can be usedto generate a composite promoter) for use in the methods of the presentinvention. The invention is not limited to the use of phage RNApolymerase promoter regions, in particular, and RNA polymerase promoterregions, in general. Suitably specific, well characterized promoters arealso found in both prokaryotic and eukaryotic systems.

[0528] The RNA that is produced in a manner that is dependent of thesuccessful detection of the target nucleic acid in the invasive cleavagereaction may be detected in any of several ways. If a labeled nucleotideis incorporated into the RNA during transcription, the RNA may bedetected directly after fractionation (e.g., by electrophoresis orchromatography). The labeled RNA may also be captured onto a solidsupport, such as a microtitre plate, a bead or a dipstick (e.g., byhybridization, antibody capture, or through an affinity interaction suchas that between biotin and avidin). Capture may facilitate the measuringof incorporated label, or it may be an intermediate step before probehybridization or similar detection means. If the maximum amount of labelis desired to be incorporated into each transcript, it is preferred thatthe copy template be very long, around 3 to 10 kilobases, so that eachRNA molecule will carry many labels. Alternatively, it may be desiredthat a single site or a limited number of sites within the transcript bespecifically labeled. In this case, it may be desirable to have a shortcopy template with only one or a few residues that would allowincorporation of the labeled nucleotide.

[0529] The copy template may also be selected to produce RNAs thatperform specified functions. In a simple case, if an duplex-dependentintercalating fluorophore is to be used to detect the RNA product, itmay be desirable to transcribe an RNA that is known to form duplexedsecondary structures, such as a ribosomal RNA or a tRNA. In anotherembodiment, the RNA may be designed to interact specifically, or withparticular affinity, with a different substance. It has been shown thata process of alternating steps of selection (e.g., by binding to atarget substance) and in vitro amplification (e.g., by PCR) can be usedto identify nucleic acid ligands with novel and useful properties (Tuerkand Gold, Science 249:505 [1990]). This system has been used to identifyRNAs, termed ligands or aptamers, that bind tightly and specifically toproteins and to other types of molecules, such as antibiotics (Wang etal., Biochem. 35:12338 [1996]) and hormones. RNAs can even be selectedto bind to other RNAs through non-Watson-Crick interactions (Schmidt etal., Ann. N.Y. Acad. Sci. 782:526 [1996]). A ligand RNA may be used toeither inactivate or enhance the activity of a molecule to which itbinds. Any RNA segment identified through such a process may also beproduced by the methods of the present invention, so that theobservation of the activity of the RNA ligand may be used as a specificsign of the presence of the target material in the invasive cleavagereaction. The ligand binding to its specific partner may also be used asanother way of capturing a readout signal to a solid support.

[0530] The product RNA might also be designed to have a catalyticfunction (e.g., to act as a ribozyme), allowing cleavage anothermolecule to be indicative of the success of the primary invasivecleavage reaction (Uhlenbeck, Nature 328:596 [1987]). In yet anotherembodiment, the RNA may be made to encode a peptide sequence. Whencoupled to an in vitro translation system (e.g., the S-30 system derivedfrom E. coli [Lesley, Methods Mol. Biol., 37:265 (1985)], or a rabbitreticulocyte lysate system [Dasso and Jackson, Nucleic Acids Res.17:3129 (1989)], available from Promega), the production of theappropriate protein may be detected. In a preferred embodiment, theproteins produced include those that allow either calorimetric orluminescent detection, such as beta-galactosidase (lac-Z) or luciferase,respectively.

[0531] The above discussion focused on the use of the presenttranscription visualization methods in the context of theINVADER-directed cleavage assay (i.e., the non-target cleavage productsproduced in the INVADER assay were used to complete and activate aprotein binding region, such as a promoter region). However, thetranscription visualization methods are not limited to this context. Anyassay that produces an oligonucleotide product having relativelydiscrete ends can be used in conjunction with the present transcriptionvisualization methods. For example, the homogenous assay described inU.S. Pat. No. 5,210,015, particularly when conducted under conditionswhere polymerization cannot occur, produces short oligonucleotidefragments as the result of cleavage of a probe. If this assay isconducted under conditions where polymerization occurs, the site ofcleavage of the probe may be focused through the use of nucleotideanalogs that have uncleavable linkages at particular positions withinthe probe. These short oligonucleotides can be employed in a manneranalogous to the cut probe or non-target cleavage products produced inthe invasive cleavage reactions of the present invention. Additionalassays that generate suitable oligonucleotide products are known to theart. For example, the non-target cleavage products produced in assayssuch as the “Cycling Probe Reaction” (Duck et al., BioTech., 9:142[1990] and U.S. Pat. Nos. 4,876,187 and 5,011,769, herein incorporatedby reference), in which shorter oligonucleotides are released fromlonger oligonucleotides after hybridization to a target sequence wouldbe suitable, as would short restriction fragments released in assayswhere a probe is designed to be cleaved when successfully hybridized toan appropriate restriction recognition sequence (U.S. Pat. No.4,683,194, herein incorporated by reference).

[0532] Assays that generate short oligonucleotides having “ragged”(i.e., not discrete) 3′ ends can also be employed with success in thetranscription reactions of the present invention when theoligonucleotide provided by this non-transcription reaction are used toprovide a portion of the promoter region located downstream of the otheroligonucleotide(s) that are required to complete the promoter region(that is a 3′ tail or unpaired extension can be tolerated when theoligonucleotide is being used as the “Cut Probe” is in FIGS. 94 and95A).

[0533] VII. Generation of 5′ Nucleases Derived From Thermostable DNAPolymerases

[0534] The 5′ nucleases of the invention form the basis of a noveldetection assay for the identification of specific nucleic acidsequences. FIG. 1A provides a schematic of one embodiment of thedetection method of the present invention. The target sequence isrecognized by two distinct oligonucleotides in the triggering or triggerreaction. In a preferred embodiment, one of these oligonucleotides isprovided on a solid support. The other can be provided free in solution.In FIG. 1A the free oligonucleotide is indicated as a “primer” and theother oligonucleotide is shown attached to a bead designated as type 1.The target nucleic acid aligns the two oligonucleotides for specificcleavage of the 5′ arm (of the oligonucleotide on bead 1) by the 5′nucleases of the present invention (not shown in FIG. 1A). The site ofcleavage (indicated by a large solid arrowhead) is controlled by theposition of the 3′ end of the “primer” relative to the downstream forkof the oligonucleotide on bead 1.

[0535] Successful cleavage releases a single copy of what is referred toas the alpha signal oligonucleotide. This oligonucleotide may contain adetectable moiety (e.g., fluorescein). On the other hand, it may beunlabeled.

[0536] In one embodiment of the detection method, two moreoligonucleotides are provided on solid supports. The oligonucleotideshown in FIG. 1A on bead 2 has a region that is complementary to thealpha signal oligonucleotide (indicated as alpha prime) allowing forhybridization. This structure can be cleaved by the 5′ nucleases of thepresent invention to release the beta signal oligonucleotide. The betasignal oligonucleotide can then hybridize to type 3 beads having anoligonucleotide with a complementary region (indicated as beta prime).Again, this structure can be cleaved by the 5′ nucleases of the presentinvention to release a new alpha oligonucleotide.

[0537] Up to this point, the amplification has been linear. To increasethe power of the method, it is desired that the alpha signaloligonucleotide hybridized to bead type 2 be liberated after release ofthe beta oligonucleotide so that it may go on to hybridize with otheroligonucleotides on type 2 beads. Similarly, after release of an alphaoligonucleotide from type 3 beads, it is desired that the betaoligonucleotide be liberated.

[0538] With the liberation of signal oligonucleotides by suchtechniques, each cleavage results in a doubling of the number of signaloligonucleotides. In this manner, detectable signal can quickly beachieved.

[0539]FIG. 1B provides a schematic of a second embodiment of thedetection method of the present invention. Again, the target sequence isrecognized by two distinct oligonucleotides in the triggering or triggerreaction and the target nucleic acid aligns the two oligonucleotides forspecific cleavage of the 5′ arm by the DNAPs of the present invention(not shown in FIG. 1B). In this specific example, the firstoligonucleotide is completely complementary to a portion of the targetsequence. The second oligonucleotide is partially complementary to thetarget sequence; the 3′ end of the second oligonucleotide is fullycomplementary to the target sequence while the 5′ end isnon-complementary and forms a single-stranded arm. The non-complementaryend of the second oligonucleotide may be a generic sequence that can beused with a set of standard hairpin structures (described below). Thedetection of different target sequences would require unique portions oftwo oligonucleotides: the entire first oligonucleotide and the 3′ end ofthe second oligonucleotide. The 5′ arm of the second oligonucleotide canbe invariant or generic in sequence.

[0540] The second part of the detection method allows the annealing ofthe fragment of the second oligonucleotide liberated by the cleavage ofthe first cleavage structure formed in the triggering reaction (calledthe third or trigger oligonucleotide) to a first hairpin structure. Thisfirst hairpin structure has a single-stranded 5′ arm and asingle-stranded 3′ arm. The third oligonucleotide triggers the cleavageof this first hairpin structure by annealing to the 3′ arm of thehairpin thereby forming a substrate for cleavage by the 5′ nuclease ofthe present invention. The cleavage of this first hairpin structuregenerates two reaction products: 1) the cleaved 5′ arm of the hairpincalled the fourth oligonucleotide, and 2) the cleaved hairpin structurethat now lacks the 5′ arm and is smaller in size than the uncleavedhairpin. This cleaved first hairpin may be used as a detection moleculeto indicate that cleavage directed by the trigger or thirdoligonucleotide occurred. Thus, this indicates that the first twooligonucleotides found and annealed to the target sequence therebyindicating the presence of the target sequence in the sample.

[0541] The detection products may be amplified by having the fourtholigonucleotide anneal to a second hairpin structure. This hairpinstructure has a 5′ single-stranded arm and a 3′ single-stranded arm. Thefourth oligonucleotide generated by cleavage of the first hairpinstructure anneals to the 3′ arm of the second hairpin structure therebycreating a third cleavage structure recognized by the 5′ nuclease. Thecleavage of this second hairpin structure also generates two reactionproducts: 1) the cleaved 5′ arm of the hairpin called the fiftholigonucleotide, and 2) the cleaved second hairpin structure which nowlacks the 5′ arm and is smaller in size than the uncleaved hairpin. Inone embodiment, the fifth oligonucleotide is similar or identical insequence to the third nucleotide. The cleaved second hairpin may beviewed as a detection molecule that amplifies the signal generated bythe cleavage of the first hairpin structure. Simultaneously with theannealing of the forth oligonucleotide, the third oligonucleotide isdissociated from the cleaved first hairpin molecule so that it is freeto anneal to a new copy of the first hairpin structure. Thedisassociation of the oligonucleotides from the hairpin structures maybe accomplished by heating or other means suitable to disruptbase-pairing interactions. As described above, conditions may beselected that allow the association and disassociation of hybridizedoligonucleotides without temperature cycling.

[0542] If fifth oligonucleotide is similar or identical in sequence tothe third oligonucleotide, further amplification of the detection signalis achieved by annealing the fifth oligonucleotide to another moleculeof the first hairpin structure. Cleavage is then performed and theoligonucleotide that is liberated then is annealed to another moleculeof the second hairpin structure. Successive rounds of annealing andcleavage of the first and second hairpin structures, provided in excess,are performed to generate a sufficient amount of cleaved hairpinproducts to be detected.

[0543] As discussed above for other embodiments of detection usingstructure-specific nuclease cleavage, any method known in the art foranalysis of nucleic acids, nucleic acid fragments or oligonucleotidesmay be applied to the detection of these cleavage products.

[0544] The hairpin structures may be attached to a solid support, suchas an agarose, styrene or magnetic bead, via the 3′ end of the hairpin.A spacer molecule may be placed between the 3′ end of the hairpin andthe bead, if so desired. The advantage of attaching the hairpinstructures to a solid support is that this prevents the hybridization ofthe two hairpin structures to one another over regions which arecomplementary. If the hairpin structures anneal to one another, thiswould reduce the amount of hairpins available for hybridization to theprimers released during the cleavage reactions. If the hairpinstructures are attached to a solid support, then additional methods ofdetection of the products of the cleavage reaction may be employed.These methods include, but are not limited to, the measurement of thereleased single-stranded 5′ arm when the 5′ arm contains a label at the5′ terminus. This label may be radioactive, fluorescent, biotinylated,etc. If the hairpin structure is not cleaved, the 5′ label will remainattached to the solid support. If cleavage occurs, the 5′ label will bereleased from the solid support.

[0545] The 3′ end of the hairpin molecule may be blocked through the useof dideoxynucleotides. A 3′ terminus containing a dideoxynucleotide isunavailable to participate in reactions with certain DNA modifyingenzymes, such as terminal transferase. Cleavage of the hairpin having a3′ terminal dideoxynucleotide generates a new, unblocked 3′ terminus atthe site of cleavage. This new 3′ end has a free hydroxyl group that caninteract with terminal transferase thus providing another means ofdetecting the cleavage products.

[0546] The hairpin structures are designed so that theirself-complementary regions are very short (generally in the range of 3-8base pairs). Thus, the hairpin structures are not stable at the hightemperatures at which this reaction is performed (generally in the rangeof 50-75° C.) unless the hairpin is stabilized by the presence of theannealed oligonucleotide on the 3′ arm of the hairpin. This instabilityprevents the polymerase from cleaving the hairpin structure in theabsence of an associated primer thereby preventing false positiveresults due to non-oligonucleotide directed cleavage.

[0547] VIII. Improved Enzymes for Use in INVADEROligonucleotide-Directed Cleavage Reactions

[0548] A cleavage structure is defined herein as a structure that isformed by the interaction of a probe oligonucleotide and a targetnucleic acid to form a duplex, the resulting structure being cleavableby a cleavage agent, including but not limited to an enzyme. Thecleavage structure is further defined as a substrate for specificcleavage by the cleavage means in contrast to a nucleic acid moleculethat is a substrate for nonspecific cleavage by agents such asphosphodiesterases. Examples of some possible cleavage structures areshown in FIG. 15. In considering improvements to enzymatic cleavageagents, one may consider the action of said enzymes on any of thesestructures, and on any other structures that fall within the definitionof a cleavage structure. The cleavage sites indicated on the structuresin FIG. 15 are presented by way of example. Specific cleavage at anysite within such a structure is contemplated.

[0549] Improvements in an enzyme may be an increased or decreased rateof cleavage of one or more types of structures. Improvements may alsoresult in more or fewer sites of cleavage on one or more of saidcleavage structures. In developing a library of new structure-specificnucleases for use in nucleic acid cleavage assays, improvements may havemany different embodiments, each related to the specific substratestructure used in a particular assay.

[0550] As an example, one embodiment of the INVADERoligonucleotide-directed cleavage assay of the present invention may beconsidered. In the INVADER oligonucleotide-directed cleavage assay, theaccumulation of cleaved material is influenced by several features ofthe enzyme behavior. Not surprisingly, the turnover rate, or the numberof structures that can be cleaved by a single enzyme molecule in a setamount of time, is very important in determining the amount of materialprocessed during the course of an assay reaction. If an enzyme takes along time to recognize a substrate (e.g., if it is presented with aless-than-optimal structure), or if it takes a long time to executecleavage, the rate of product accumulation is lower than if these stepsproceeded quickly. If these steps are quick, yet the enzyme “holds on”to the cleaved structure, and does not immediately proceed to anotheruncut structure, the rate will be negatively affected.

[0551] Enzyme turnover is not the only way in which enzyme behavior cannegatively affect the rate of accumulation of product. When the meansused to visualize or measure product is specific for a precisely definedproduct, products that deviate from that definition may escapedetection, and thus the rate of product accumulation may appear to belower than it is. For example, if one had a sensitive detector fortrinucleotides that could not see di- or tetranucleotides, or any sizedoligonucleotide other that 3 residues, in the INVADER-directed cleavageassay of the present invention any errant cleavage would reduce thedetectable signal proportionally. It can be seen from the cleavage datapresented here that, while there is usually one site within a probe thatis favored for cleavage, there are often products that arise fromcleavage one or more nucleotides away from the primary cleavage site.These are products that are target-dependent, and are thus notnon-specific background. Nevertheless, if a subsequent visualizationsystem can detect only the primary product, these represent a loss ofsignal. One example of such a selective visualization system is thecharge reversal readout presented herein, in which the balance ofpositive and negative charges determines the behavior of the products.In such a system the presence of an extra nucleotide or the absence ofan expected nucleotide can excluded a legitimate cleavage product fromultimate detection by leaving that product with the wrong balance ofcharge. It can be easily seen that any assay that can sensitivelydistinguish the nucleotide content of an oligonucleotide, such asstandard stringent hybridization, suffers in sensitivity when somefraction of the legitimate product is not eligible for successfuldetection by that assay.

[0552] These discussions suggest two highly desirable traits in anyenzyme to be used in the method of the present invention. First, themore rapidly the enzyme executes an entire cleavage reaction, includingrecognition, cleavage and release, the more signal it may potentiallycreated in the INVADER oligonucleotide-directed cleavage assay. Second,the more successful an enzyme is at focusing on a single cleavage sitewithin a structure, the more of the cleavage product can be successfullydetected in a selective read-out.

[0553] The rationale cited above for making improvements in enzymes tobe used in the INVADER oligonucleotide-directed cleavage assay are meantto serve as an example of one direction in which improvements might besought, but not as a limit on either the nature or the applications ofimproved enzyme activities. As another direction of activity change thatwould be appropriately considered improvement, the DNAP-associated 5′nucleases may be used as an example. In creating some of thepolymerase-deficient 5′ nucleases described herein it was found that thethose that were created by deletion of substantial portions of thepolymerase domain, as depicted in FIG. 4, assumed activities that wereweak or absent in the parent proteins. These activities included theability to cleave the non-forked structure shown in FIG. 15D, a greatlyenhanced ability to exonucleolytically remove nucleotides from the 5′ends of duplexed strands, and a nascent ability to cleave circularmolecules without benefit of a free 5′ end.

[0554] In addition to the 5′ nucleases derived from DNA polymerases, thepresent invention also contemplates the use of structure-specificnucleases that are not derived from DNA polymerases. For example, aclass of eukaryotic and archaebacterial endonucleases have beenidentified which have a similar substrate specificity to 5′ nucleases ofPol I-type DNA polymerases. These are the FEN1 (Flap EndoNuclease),RAD2, and XPG (Xeroderma Pigmentosa-complementation group G) proteins.These proteins are involved in DNA repair, and have been shown to favorthe cleavage of structures that resemble a 5′ arm that has beendisplaced by an extending primer during polymerization, similar to themodel depicted in FIG. 15B. Similar DNA repair enzymes have beenisolated from single cell and higher eukaryotes and from archaea, andthere are related DNA repair proteins in eubacteria. Similar 5′nucleases have also been associated with bacteriophage such as T5 andT7.

[0555] Recently, the 3-dimensional structures of DNAPTaq and T5 phage5′-exonuclease (FIG. 58) were determined by X-ray diffraction (Kim etal., Nature 376:612 [1995]; and Ceska et al., Nature 382:90 [1995]). Thetwo enzymes have very similar 3-dimensional structures despite limitedamino acid sequence similarity. The most striking feature of the T55′-exonuclease structure is the existence of a triangular hole formed bythe active site of the protein and two alpha helices (FIG. 58). Thissame region of DNAPTaq is disordered in the crystal structure,indicating that this region is flexible, and thus is not shown in thepublished 3-dimensional structure. However, the 5′ nuclease domain ofDNAPTaq is likely to have the same structure, based its overall3-dimensional similarity to T5 5′-exonuclease, and that the amino acidsin the disordered region of the DNAPTaq protein are those associatedwith alpha helix formation. The existence of such a hole or groove inthe 5′ nuclease domain of DNAPTaq was predicted based on its substratespecificity (Lyamichev et al., supra).

[0556] It has been suggested that the 5′ arm of a cleavage structuremust thread through the helical arch described above to position saidstructure correctly for cleavage (Ceska et al., supra). One of themodifications of 5′ nucleases described herein opened up the helicalarch portion of the protein to allow improved cleavage of structuresthat cut poorly or not at all (e.g., structures on circular DNA targetsthat would preclude such threading of a 5′ arm). The gene construct thatwas chosen as a model to test this approach was the one called CLEAVASEBN, which was derived from DNAPTaq but does not contain the polymerasedomain (Ex. 2). It comprises the entire 5′ nuclease domain of DNAP Taq,and thus should be very close in structure to the T5 5′ exonuclease.This 5′ nuclease was chosen to demonstrate the principle of such aphysical modification on proteins of this type. The arch-openingmodification of the present invention is not intended to be limited tothe 5′ nuclease domains of DNA polymerases, and is contemplated for useon any structure-specific nuclease that includes such an aperture as alimitation on cleavage activity. The present invention contemplates theinsertion of a thrombin cleavage site into the helical arch of DNAPsderived from the genus Thermus as well as 5′ nucleases derived fromDNAPs derived from the genus Thermus. The specific example shown hereinusing the CLEAVASE BN/thrombin nuclease merely illustrates the conceptof opening the helical arch located within a nuclease domain. As theamino acid sequence of DNAPs derived from the genus Thermus are highlyconserved, the teachings of the present invention enable the insertionof a thrombin site into the helical arch present in these DNAPs and 5′nucleases derived from these DNAPs.

[0557] The opening of the helical arch was accomplished by insertion ofa protease site in the arch. This allowed post-translational digestionof the expressed protein with the appropriate protease to open the archat its apex. Proteases of this type recognize short stretches ofspecific amino acid sequence. Such proteases include thrombin and factorXa. Cleavage of a protein with such a protease depends on both thepresence of that site in the amino acid sequence of the protein and theaccessibility of that site on the folded intact protein. Even with acrystal structure it can be difficult to predict the susceptibility ofany particular region of a protein to protease cleavage. Absent acrystal structure it must be determined empirically.

[0558] In selecting a protease for a site-specific cleavage of a proteinthat has been modified to contain a protease cleavage site, a first stepis to test the unmodified protein for cleavage at alternative sites. Forexample, DNAPTaq and CLEAVASE BN nuclease were both incubated underprotease cleavage conditions with factor Xa and thrombin proteases. Bothnuclease proteins were cut with factor Xa within the 5′ nuclease domain,but neither nuclease was digested with large amounts of thrombin. Thus,thrombin was chosen for initial tests on opening the arch of theCLEAVASE BN enzyme.

[0559] In the protease/CLEAVASE modifications described herein thefactor Xa protease cleaved strongly in an unacceptable position in theunmodified nuclease protein, in a region likely to compromise theactivity of the end product. Other unmodified nucleases contemplatedherein may not be sensitive to the factor Xa, but may be sensitive tothrombin or other such proteases. Alternatively, they may be sensitiveto these or other such proteases at sites that are immaterial to thefunction of the nuclease sought to be modified. In approaching anyprotein for modification by addition of a protease cleavage site, theunmodified protein should be tested with the proteases underconsideration to determine which proteases give acceptable levels ofcleavage in other regions.

[0560] Working with the cloned segment of DNAPTaq from which theCLEAVASE BN protein is expressed, nucleotides encoding a thrombincleavage site were introduced in-frame near the sequence encoding aminoacid 90 of the nuclease gene. This position was determined to be at ornear the apex of the helical arch by reference to both the 3-dimensionalstructure of DNAPTaq, and the structure of T5 5′ exonuclease. Theencoded amino acid sequence, LVPRGS, was inserted into the apex of thehelical arch by site-directed mutagenesis of the nuclease gene. Theproline (P) in the thrombin cleavage site was positioned to replace aproline normally in this position in CLEAVASE BN because proline is analpha helix-breaking amino acid, and may be important for the3-dimensional structure of this arch. This construct was expressed,purified and then digested with thrombin. The digested enzyme was testedfor its ability to cleave a target nucleic acid, bacteriophage M13genomic DNA, that does not provide free 5′ ends to facilitate cleavageby the threading model.

[0561] While the helical arch in this nuclease was opened by proteasecleavage, it is contemplated that a number of other techniques could beused to achieve the same end. For example, the nucleotide sequence couldbe rearranged such that, upon expression, the resulting protein would beconfigured so that the top of the helical arch (amino acid 90) would beat the amino terminus of the protein, the natural carboxyl and aminotermini of the protein sequence would be joined, and the new carboxylterminus would lie at natural amino acid 89. This approach has thebenefit that no foreign sequences are introduced and the enzyme is asingle amino acid chain, and thus may be more stable that the cleaved 5′nuclease. In the crystal structure of DNAPTaq, the amino and carboxyltermini of the 5′-exonuclease domain lie in close proximity to eachother, which suggests that the ends may be directly joined without theuse of a flexible linker peptide sequence as is sometimes necessary.Such a rearrangement of the gene, with subsequent cloning and expressioncould be accomplished by standard PCR recombination and cloningtechniques known to those skilled in the art.

[0562] The present invention also contemplates the use of nucleasesisolated from organisms that grow under a variety of conditions. Thegenes for the FEN-1/XPG class of enzymes are found in organisms rangingfrom bacteriophage to humans to the extreme thermophiles of KingdomArchaea. For assays in which high temperature is to be used, it iscontemplated that enzymes isolated from extreme thermophiles may exhibitthe thermostability required of such an assay. For assays in which itmight be desirable to have peak enzyme activity at moderate temperatureor in which it might be desirable to destroy the enzyme with elevatedtemperature, those enzymes from organisms that favor moderatetemperatures for growth may be of particular value.

[0563] An alignment of a collection of FEN-1 proteins sequenced byothers is shown in FIGS. 59A-E (SEQ ID NOS: 135-145). It can be seenfrom this alignment that there are some regions of conservation in thisclass of proteins, suggesting that they are related in function, andpossibly in structure. Regions of similarity at the amino acid sequencelevel can be used to design primers for in vitro amplification (PCR) bya process of back translating the amino acid sequence to the possiblenucleic acid sequences, then choosing primers with the fewest possiblevariations within the sequences. These can be used in low stringency PCRto search for related DNA sequences. This approach permits theamplification of DNA encoding a FEN-1 nuclease without advance knowledgeof the actual DNA sequence.

[0564] It can also be seen from this alignment that there are regions inthe sequences that are not completely conserved. The degree ofdifference observed suggests that the proteins may have subtle ordistinct differences is substrate specificity. In other words, they mayhave different levels of cleavage activity on the cleavage structures ofthe present invention. When a particular structure is cleaved at ahigher rate than the others, this is referred to a preferred substrate,while a structure that is cleaved slowly is considered a less preferredsubstrate. The designation of preferred or less preferred substrates inthis context is not intended to be a limitation of the presentinvention. It is contemplated that some embodiments the presentinvention will make use of the interactions of an enzyme with a lesspreferred substrate. Candidate enzymes are tested for suitability in thecleavage assays of the present invention using the assays describedbelow.

[0565] 1. Structure Specific Nuclease Assay

[0566] Testing candidate nucleases for structure-specific activities inthese assays is done in much the same way as described for testingmodified DNA polymerases in Example 2, but with the use of a differentlibrary of model structures. In addition to assessing the enzymeperformance in primer-independent and primer-directed cleavage, a set ofsynthetic hairpins are used to examine the length of duplex downstreamof the cleavage site preferred by the enzyme.

[0567] The FEN-1 and XPG 5′ nucleases used in the present inventionshould be tested for activity in the assays in which they are intendedto be used, including but not limited to the INVADER-directed cleavagedetection assay of the present invention and the CFLP method ofcharacterizing nucleic acids (the CFLP method is described in U.S. Pat.Nos. 5,843,654, 5,843,669, 5,719,028, and 5,888,780 and PCT PublicationWO 96/15267; the disclosures of which are incorporated herein byreference). The INVADER assay uses a mode of cleavage that has beentermed “primer directed” of “primer dependent” to reflect the influenceof the an oligonucleotide hybridized to the target nucleic acid upstreamof the cleavage site. In contrast, the CFLP reaction is based on thecleavage of folded structure, or hairpins, within the target nucleicacid, in the absence of any hybridized oligonucleotide. The testsdescribed herein are not intended to be limited to the analysis ofnucleases with any particular site of cleavage or mode of recognition ofsubstrate structures. It is contemplated that enzymes may be describedas 3′ nucleases, utilizing the 3′ end as a reference point to recognizestructures, or may have a yet a different mode of recognition. Further,the use of the term 5′ nucleases is not intended to limit considerationto enzymes that cleave the cleavage structures at any particular site.It refers to a general class of enzymes that require some reference oraccess to a 5′ end to effect cleavage of a structure.

[0568] A set of model cleavage structures have been created to allow thecleavage ability of unknown enzymes on such structures to be assessed.Each of the model structures is constructed of one or more syntheticoligonucleotides made by standard DNA synthesis chemistry. Examples ofsuch synthetic model substrate structures are shown in FIGS. 26 and 60.These are intended only to represent the general folded configurationdesirable is such test structures. While a sequence that would assumesuch a structure is indicated in the Figures, there are numerous othersequence arrangements of nucleotides that would be expected to fold insuch ways. The essential features to be designed into a set ofoligonucleotides to perform the tests described herein are the presenceor absence of a sufficiently long 3′ arm to allow hybridization of anadditional nucleic acid to test cleavage in a “primer-directed” mode,and the length of the duplex region. In the set depicted in FIG. 60, theduplex lengths of the S-33 and the 11-8-0 structures are 12 and 8basepairs, respectively. This difference in length in the test moleculesfacilitates detection of discrimination by the candidate nucleasebetween longer and shorter duplexes. Additions to this series expandingthe range of duplex molecules presented to the enzymes, both shorter andlonger, may be used. The use of a stabilizing DNA tetraloop (Antao etal., Nucl. Acids Res., 19:5901 [1991]) or triloop (Hiraro et al., Nuc.Acids Res., 22:576 [1994]) at the closed end of the duplex helps ensureformation of the expected structure by the oligonucleotide.

[0569] The model substrate for testing primer directed cleavage, the“S-60 hairpin” (SEQ ID NO: 40) is described in Example 11. In theabsence of a primer this hairpin is usually cleaved to release 5′ armfragments of 18 and 19 nucleotides length. An oligonucleotide, termedP-14 (5′-CGAGAGACCACGCT-3′; SEQ ID NO: 108), that extends to the base ofthe duplex when hybridized to the 3′ arm of the S-60 hairpin givescleavage products of the same size, but at a higher rate of cleavage.

[0570] To test invasive cleavage a different primer is used, termed P-15(5′-CGAGAGACCACGCTG-3′; SEQ ID NO: 30). In a successful invasivecleavage the presence of this primer shifts the site of cleavage of S-60into the duplex region, usually releasing products of 21 and 22nucleotides length.

[0571] The S-60 hairpin may also be used to test the effects ofmodifications of the cleavage structure on either primer-directed orinvasive cleavage. Such modifications include, but are not limited to,use of mismatches or base analogs in the hairpin duplex at one, a few orall positions, similar disruptions or modifications in the duplexbetween the primer and the 3′ arm of the S-60, chemical or othermodifications to one or both ends of the primer sequence, or attachmentof moieties to, or other modifications of the 5′ arm of the structure.In all of the analyses using the S-60 or a similar hairpin describedherein, activity with and without a primer may be compared using thesame hairpin structure.

[0572] The assembly of these test reactions, including appropriateamounts of hairpin, primer and candidate nuclease are described inExample 2. As cited therein, the presence of cleavage products isindicated by the presence of molecules which migrate at a lowermolecular weight than does the uncleaved test structure. When thereversal of charge of a label is used the products will carry adifferent net charge than the uncleaved material. Any of these cleavageproducts indicate that the candidate nuclease has the desiredstructure-specific nuclease activity. By “desired structure-specificnuclease activity” it is meant only that the candidate nuclease cleavesone or more test molecules. It is not necessary that the candidatenuclease cleave at any particular rate or site of cleavage to beconsidered successful cleavage.

[0573] IX. The INVADER Assay for Direct Detection and Measurement ofSpecific Analytes

[0574] The following description provides illustrative examples oftarget sequence detection through the use of the compositions andmethods of the present invention. These example include the detection ofhuman cytomegalovirus viral DNA, single nucleotide polymorphisms in thehuman apolipoprotein E gene, mutations in the human hemochromatosisgene, mutations in the human MTHFR, prothrombin 20210GA polymorphism,the HR-2 mutation in the human Factor V gene, single nucleotidepolymorphisms in the human TNF-α Gene, and Leiden mutation in the humanFactor V gene. Included in these descriptions are novel nucleic acidcompositions for use in the detection of such sequence. Examples 54-61below provide details on the design and execution of these illustrativeembodiments.

[0575] A. Detection of Human Cytomegalovirus Viral DNA by InvasiveCleavage

[0576] Human cytomegalovirus (HCMV) causes, or is associated with, awide variety of diseases in humans (Table 3). More than 90% of bonemarrow or kidney transplant recipients (immunocompromised hosts) developHCMV infections, most of which are due to reactivation of latent virusby immunosuppressive drugs, as well as transmission of virus by latentlyinfected donor tissue or blood (Ackerman et al., Transplant. Proc.,20(S1):468 [1988]; and Peterson et al., Medicine 59:283 [1980]). TABLE 3Diseases Caused By Human Cytomegalovirus cytomegalic inclusionheterophil-negative disease in neonates mononucleosis interstitialpneumonia pneumonitis retinitis hepatitis pancreatitismeningoencephalitis gastrointestinal disease disseminated infection

[0577] There are instances in which rapid, sensitive, and specificdiagnosis of HCMV disease is imperative. In recent years, the number ofpatients undergoing organ and tissue transplantations has increasedmarkedly. HCMV is the most frequent cause of death in immunocompromisedtransplant recipients, thereby confirming the need for rapid andreliable laboratory diagnosis. Lymphocytes, monocytes, and possiblyarterial endothelial or smooth muscle cells, are sites of HCMV latency.Therefore, prevention of HCMV infections in immunocompromisedindividuals (e.g., transplant recipients) includes use of HCMV-negativeblood products and organs. Additionally, HCMV can be spreadtransplacentally, and to newborns by contact with infected cervicalsecretions during birth. Thus, a rapid, sensitive, and specific assayfor detecting HCMV in body fluids or secretions may be desirable as ameans to monitor infection, and consequently, determine the necessity ofcesarean section.

[0578] Diagnosis of HCMV infection may be performed by conventional cellculture using human fibroblasts; shell vial centrifugation cultureutilizing monoclonal antibodies and immunofluorescent stainingtechniques; serological methods; the HCMV antigenemia assay whichemploys a monoclonal antibody to detect HCMV antigen in peripheral bloodleukocytes (PBLs); or by nucleic acid hybridization assays. Thesevarious methods have their advantages and limitations. Conventional cellculture is sensitive but slow, as cytopathic effect (CPE) may take 30 ormore days to develop. Shell vial centrifugation is more rapid but stillrequires 24-48 hours for initial results. Both culture methods areaffected by antiviral therapy. In immunocompromised patients, theability to mount IgG and/or IgM antibody responses to HCMV infection areimpaired, and serological methods are thus not reliable in this setting.Alternatively, IgM antibodies may be persistent for months afterinfection is resolved, and thus their presence may not be indicative ofactive infection. The HCMV antigenemia assay is labor intensive and isnot applicable to specimens other than PBLs.

[0579] Recent advances in molecular biology have spurred the use of DNAprobes in attempts to provide a more rapid, sensitive and specific assayfor detecting HCMV in clinical specimens. For example, radiolabeled DNAprobes have been used to hybridize to tissue cultures infected with orby HCMV, or in clinical samples suspected of containing HCMV(“hybridization assays”). However, probing of tissue cultures requiresat least 18-24 hours for growth to amplify the antigen (HCMV) to bedetected, if present, and additional time for development ofautoradiographic detection systems. Using hybridization assays forassaying clinical specimens for HCMV may lack sensitivity, dependingupon the titer of virus and the clinical sample assayed. Detection ofHCMV in clinical samples has been reported using the polymerase chainreaction (PCR) to enzymatically amplify HCMV DNA. Methods using PCRcompare favorably with virus isolation, in situ hybridization assays,and Southern blotting; See, e.g., Bamborschke et al,. J. Neurol.,239:205 [1992]; Drouet et al., J. Virol. Meth., 45:259 [1993]; Einseleet al., Blood 77:1104-1110 [1991]; Einsele et al., Lancet 338:1170[1991]; Lee et al., Aust. NZ J. Med., 22:249 [1992]; Miller et al., J.Clin. Microbiol., 32:5 [1994]; Rowley et al., Transplant. 51:1028[1991]; Spector et al. J. Clin. Microbiol., 30:2359 [1992]; and Stanieret al., Mol. Cell. Probes 8:51 [1992]). Others, comparing the HCMVantigenemia assay with PCR methods, have found PCR methods as efficientor slightly more efficient in the detection of HCMV (van Dorp et al.(1992) Transplant. 54:661; Gema et al. (1991) J. Infect. Dis. 164:488;Vleiger et al. (1992) Bone Marrow Transplant. 9:247; Zipeto et al.(1992) J. Clin. Microbiol. 30:527]. In addition, PCR methods haveexhibited great sensitivity when specimens other than PBLs are assayed(Natori et al., Kansenshogaku Zasshi 67:1011 [1993]; Peterson et al.,Medicine 59:283 [1980]; Prosch et al., J. Med. Virol., 38:246 [1992];Ratnamohan et al., J. Med. Virol. 38:252 [1992]). However, because ofthe dangers of false positive reactions, these PCR-based proceduresrequire rigid controls to prevent contamination and carry over (Ehrlichet al., in PCR-Based Diagnostics in Infectious Diseases, Ehrlich andGreenberg (eds), Blackwell Scientific Publications, [1994], pp.3-18).Therefore, there exists a need for a rapid, sensitive, and specificassay for HCMV that has a reduced risk of false positive result due tocontamination by reaction product carried over from other samples.

[0580] As shown herein, the INVADER-directed cleavage assay is rapid,sensitive and specific. Because the accumulated products do notcontribute to the further accumulation of signal, reaction productscarried over from one standard (i.e., non-sequential) INVADER-directedcleavage assay to another cannot promote false positive results. The useof multiple sequential INVADER-directed cleavage assays will furtherboost the sensitivity of HCMV detection without sacrifice of theseadvantages.

[0581] B. Detection of Single Nucleotide Polymorphisms in the HumanApolipoprotein E Gene

[0582] Apolipoprotein E (ApoE) performs various functions as a proteinconstituent of plasma lipoproteins, including its role in cholesterolmetabolism. It was first identified as a constituent ofliver-synthesized very low density lipoproteins which function in thetransport of triglycerides from the liver to peripheral tissues. Thereare three major isoforms of ApoE, referred to as ApoE2, ApoE3 and ApoE4which are products of three alleles at a single gene locus. Threehomozygous phenotypes (Apo-E2/2, E3/3, and E4/4) and three heterozygousphenotypes (ApoE3/2, E4/3 and E4/2) arise from the expression of any twoof the three alleles. The most common phenotype is ApoE3/3 and the mostcommon allele is E3. See Mahley, R. W., Science 240:622-630 (1988).

[0583] The amino acid sequences of the three types differ only slightly.ApoE4 differs from ApoE3 in that in ApoE4 arginine is substituted forthe normally occurring cysteine at amino acid residue 112. The mostcommon form of ApoE2 differs from ApoE3 at residue 158, where cysteineis substituted for the normally occurring arginine. See Mahley, Science,supra.

[0584] The frequency of the apoE4 allele has been shown to be markedlyincreased in sporadic Alzheimer's Disease (AD) (Poirier, J. et al.,1993, Apolipoprotein E phenotype and Alzheimer's Disease, Lancet,342:697-699; Noguchi, S. et al., 1993, Lancet (letter), 342:737) andlate onset familial Alzheimer's disease (AD) (Corder, E. H. et al.,1993, Science, 261:921-923; Payami, H. et al., 1993, Lancet (letter),342:738). This gene dosage effect was observed in both sporadic andfamilial cases (i.e., as age of onset increases, E4 allele copy numberdecreases). Women, who are generally at a greater risk of developingAlzheimer's disease, show increased E4 allele frequency when compared toage matched men.

[0585] C. Detection of Mutations in the Human Hemochromatosis Gene

[0586] Hereditary hemochromatosis (HH) is an inherited disorder of ironmetabolism wherein the body accumulates excess iron. In symptomaticindividuals, this excess iron leads to deleterious effects by beingdeposited in a variety of organs leading to their failure, and resultingin cirrhosis, diabetes, sterility, and other serious illnesses.

[0587] HH is inherited as a recessive trait; heterozygotes areasymptomatic and only homozygotes are affected by the disease. It isestimated that approximately 10% of individuals of Western Europeandescent carry an HH gene mutation and that there are about one millionhomozygotes in the United States. Although ultimately HH producesdebilitating symptoms, the majority of homozygotes have not beendiagnosed. Indeed, it has been estimated that no more than 10,000 peoplein the United States have been diagnosed with this condition. Thesymptoms are often confused with those of other conditions, and thesevere effects of the disease often do not appear immediately. It wouldbe desirable to provide a method to identify persons who are ultimatelydestined to become symptomatic in order to intervene in time to preventexcessive tissue damage. One reason for the lack of early diagnosis isthe inadequacy of presently available diagnostic methods to ascertainwhich individuals are at risk.

[0588] Although blood iron parameters can be used as a screening tool, aconfirmed diagnosis often employs HLA typing, which is tedious,nonspecific, and expensive and/or liver biopsy which is undesirablyinvasive and costly. Accordingly, others have attempted to developinexpensive and noninvasive diagnostics both for detection ofhomozygotes having existing disease, in that presymptomatic detectionwould guide intervention to prevent organ damage, and for identificationof carriers. The need for such diagnostics is documented for example, inFinch, C. A. West J Med (1990) 153:323-325; McCusick, V. et al.Mendelian Inheritance in Man 11th ed., Johns Hopkins University Press(Baltimore, 1994) pp. 1882-1887; Report of the Joint World HealthOrganization/HH Foundation/French HH Association Meeting, 1993.

[0589] D. Detection of Mutations in the Human MTHFR

[0590] Folic acid derivatives are coenzymes for several criticalsingle-carbon transfer reactions, including reactions in thebiosynthesis of purines, thymidylate and methionine.Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) catalyzes theNADPH-linked reduction of 5,10-methylenetetrahydrofolate to5-methyltetrahydrofolate, a co-substrate for methylation of homocysteineto methionine. The porcine liver enzyme, a flavoprotein, has beenpurified to homogeneity; it is a homodimer of 77-kDa subunits. Partialproteolysis of the porcine peptide has revealed two spatially distinctdomains: an N-terminal domain of 40 kDa and a C-terminal domain of 37kDa. The latter domain contains the binding site for the allostericregulator S-adenosylmethionine.

[0591] Hereditary deficiency of MTHFR, an autosomal recessive disorder,is the most common inborn error of folic acid metabolism. A block in theproduction of methyltetrahydrofolate leads to elevated homocysteine withlow to normal levels of methionine. Patients with severe deficiencies ofMTHFR (0-20% activity in fibroblasts) can have variable phenotypes.Developmental delay, mental retardation, motor and gait abnormalities,peripheral neuropathy, seizures and psychiatric disturbances have beenreported in this group, although at least one patient with severe MTHFRdeficiency was asymptomatic. Pathologic changes in the severe forminclude the vascular changes that have been found in other conditionswith elevated homocysteine, as well as reduced neurotransmitter andmethionine levels in the CNS. A milder deficiency of MTHFR (35-50%activity) has been described in patients with coronary artery disease.Genetic heterogeneity is likely, considering the diverse clinicalfeatures, the variable levels of enzyme activity, and the differentialheat inactivation profiles of the reductase in patients' cells. Methodsto detect the MTHFR mutation include: AS-PCR (Hessner, et al. Br JHaematol 106, 237-9 (1999)) and PCR-RFLP (Nature Genetics, Frosst etal.1995:10; 111-113).

[0592] E. Detection of Prothrombin 20210GA Polymorphism and the Factor VLeiden Polymorphism

[0593] The coagulation cascade is a complex series of zymogenactivations, inactivations and feed back loops involving numerousenzymes and their cofactors. The entire cascade, from tissue injury orvenous trauma to clotting has been well described (refs). The cascadeculminates in the conversion of prothrombin (Factor II) to thrombin.This is catalyzed by the activated form of factor X, factor Xa and itscofactor, activated factor V, factor Va. Thrombin then convertsfibrinogen to fibrin and promotes fibrin cross-linking and clotformation by activating factor XIII. In addition to the above statedfunctions, thrombin a serine protease, can also activate factor V in apositive feed-back loop. Factor Va is a pro-coagulant cofactor in theclotting cascade, and when clot formation is sufficient, is inactivatedby activated protein C (APC).

[0594] Venous thrombosis is the obstruction of the circulation by clotsthat have been formed in the veins or have been released from a thrombusformed elsewhere. The most frequent sites of clot formation are the deepveins of the legs, but it also may occur in veins in the brain, retina,liver and mesentery. Factors other than heritable defects that can playa role in the development of thrombosis include recent surgery,malignant disorders, pregnancy and labor and long term immobilization.

[0595] Studies of hereditary thrombophilia, defined as an increasedtendency towards venous thrombotic disease in relatively young adults,have provided insights into the genetic factors that regulatethrombosis. In 1993, Dahlback et al. (Proc Natl Acad Sci USA 1993;90:1004-1008) described an insensitivity to APC, a critical anti-coagulantin the clotting cascade, in three unrelated families with hereditarythrombophilia. The anticoagulant property of APC resides in its capacityto inactivate the activated cofactors Va and VIIIa by limitedproteolysis (ref 3). This inactivation of cofactors Va and VIIIa resultsin reduction of the rate of formation of thrombin, the end product ofthe cascade. This observation was confirmed by other investigators (ref)and the term “APC resistance” was coined to describe this particularphenotype in thrombophilic patients. In a subsequent study of 20families with thrombophilia and APC resistance, an autosomal dominantpattern of inheritance was observed (17). Bertina et al (Nature, 1994,May 5;369 (6475):64-7) then demonstrated that the phenotype of APCresistance is associated with heterozygosity or homozygosity for asingle point mutation at nucleotide 1691 in exon 10 of the factor Vgene. This single base change, a guanine to adenine substitution, yieldsa mutant factor V molecule wherein the arginine at position 506 isreplaced with glutamine. This form of the factor V molecule,characterized at Leiden University, (Bertenia et al) is known as the FVQ506 or FV Leiden mutation, and is inactivated less efficiently by APCthan the wild type protein. It has been postulated that the prolongedcirculation of activated factor V promotes a hypercoagulable state andincreases the risk of thrombosis. Subsequent analysis of various patientgroups exhibiting symptoms of venous thrombosis indicate that the factorV Leiden mutation is the single most common heritable factorcontributing to an increased risk of venous thrombosis.

[0596] In 1996, studies by Poort et al. (Blood. 1996:88; 3698-703)revealed the second most common heritable factor contributing toincrease thrombotic risk. In studying the sequence of the prothrombingene in selected patients with a documented familial history of venousthrombophilia, the Poort group identified a single point mutation in the3′ untranslated region. This G to A transition at position 20210 isstrongly correlated with elevated plasma prothrombin levels, and wasalso shown to be associated with an almost threefold increased risk ofvenous thrombosis (abstract, Howard)

[0597] The first reported case of a thrombophilia pateint geneticallyhomozygous for the G to A polymorphism in the 3′ untranslated region wasby Howard, et al (Blood Coagulation Fibrinolysis July 1997; 8(5):316-9).The patient, a healthy young Mexican male presented with a myocardialinfarction, venous thrombosis and embolism. The patient was found to behomozygous for the prothrombin mutation and heterozygous for the FactorV Leiden mutation, supporting the doublehit theory for thrombophilia inyoung patients.

[0598] Studies by Hessner et al. show that the prothrombin 20210GAgenotype was nearly 5 times as prevalent in the symptomoatic FVLcarriers than in a random Caucasian control group (British Journal ofHaematology, 1999, 106), and that allele frequencies for the prothrombinand Factor V mutants vary among different ethnic backgrounds (ThrombHaemostat 1999; 81:733-8). The above discussion confirms that earlydetection of the factor V Leiden mutation and the factor II prothrombinmutation are paramount in hereditary thrombotic risk assessment. Thenature of these two mutations, that is, a single base change in thenucleic acid sequence, make them amenable to a variety of nucleic aciddetection methods known to the art, though the demand for faster, morereliable, cost-effective and user-friendly tests for the detection ofspecific nucleic acid sequences continues to grow. The most commonmethods to test for these mutations include PCR/RFLP, AS-PCR andfunctional, coagulation assay.

[0599] F. Detection of the HR-2 Mutation in the Human Factor V Gene

[0600] The R-2 polymorphism is located in exon 13 of the factor V gene,and is the result of an A to G transition at base 4070, replacing thewild-type amino acid histine with the mutant argenine in the matureprotein. The R-2 polymorphism is one of a set of mutations termedcollectively HR-2. The HR-2 haplotype is defined by 6 nucleotide basesubstitutions in exons 13 and 16 of the factor V gene. The haplotype isassociated with an increased functional resistance to activated proteinC both in normal subjects and in thrombophilic patients. When present asa compound heterozygote in conjunction with the factor V Leidenmutation, clinical symptoms are comparable to those seen in patientshomozygous for the factor V Leiden mutation, and include increased riskof deep vein thrombosis.

[0601] G. Detection of Single Nucleotide Polymorphisms in the HumanTNF-αGene

[0602] The human cytokine tumor necrosis factor alpha (TNF-alpha) hasbeen shown to be a major factor in graft rejection; the more TNF-alphapresent in the system, the greater the rejection response totransplanted tissue. Mutations in TNF-alpha have also been correlatedwith cerebral malaria (Nature 1994;371:508-510), fulminas purpura (JInfect Dis. 1996;174:878-880), and mucocutaneous leishmaniaisis (J ExpMed. 1995;182:1259-1264). The mutation detected in this example islocated in the promoter region of the TNF-alpha gene at position minus308. The wild-type guanine (G) is replaced with a mutant adenine (A).This result of this promoter mutation is the enhancement oftranscription of TNF-alpha by 6-7 fold. Methods to detect mutations inTNF-alpha include sequencing, denaturing gradient gel electorphoresis,PCR methods, and methods involving both PCR and post-PCR hybridizationwith specific oligos.

[0603] H. Detection of Methicillin Resistant Staphylococcus aureus

[0604]Staphylococcus aureus is recognized as one of the major causes ofinfections in humans occurring in both in the hospital and in thecommunity at large. One of the most serious concerns in treating anybacterial infection is the increasing resistance to antibiotics. Thegrowing incidence of methicillin-resistant S. aureus (MRSA) infectionsworldwide has underscored the importance of both early detection of theinfective agent, and defining a resistance profile such that propertreatment can be given. The primary mechanism for resistance tomethicillin involves the production of a protein called PBP2a, encodedby the mecA gene. The mecA gene not specific to Staphalococcus aureus,but is of extraspecies origin. The mecA gene is however, indicative ofmethicillin resistance and is used as a marker for the detection ofresistant bacteria. So, to identify methicillin resistant S. aureus vianucleic acid techniques, both the mecA gene and at least one speciesspecific gene must be targeted. A particular species specific gene, thenuclease or nuc gene is used in the following example. Methods used todetect MRSA include time consuming and laborious culturing andcoagulation assays and growth assays on antibiotic media. Molecularapproaches include a Cycling Probe™ assay, the Velogene™ Kit fromAlexon-Trend (Ramsey, Minn. cat #818-48), anti-body test which bind thePBP2a protein, bDNA Assay (Chiron, Emeryville, Calif.), all of whichtests only for the presence of the mecA gene and are not Staph. aureusspecific.

EXAMPLES

[0605] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof

[0606] In the disclosure which follows, the following abbreviationsapply: Afu (Archaeoglobus fulgidus); Mth (Methanobacteriumthermoautotrophicum); Mja (Methanococcus jannaschii); Pfu (Pyrococcusfuriosus); Pwo (Pyrococcus woesei); Taq (Thermus aquaticus); Taq DNAP,DNAPTaq, and Taq Pol I (T. aquaticus DNA polymerase I); DNAPStf (theStoffel fragment of DNAPTaq); DNAPEcl (E. coli DNA polymerase I); Tth(Thermus thermophilus); Ex. (Example); Fig. (Figure);° C. (degreesCentigrade); g (gravitational field); hr (hour); min (minute); ollo(oligonucleotide); rxn (reaction); vol (volume); w/v (weight to volume);v/v (volume to volume); BSA (bovine serum albumin); CTAB(cetyltrimethylammonium bromide); HPLC (high pressure liquidchromatography); DNA (deoxyribonucleic acid); p (plasmid); μl(microliters); ml (milliliters); pg (micrograms); mg (milligrams); M(molar); mM (milliMolar); μM (microMolar); pmoles (picomoles); amoles(attomoles); zmoles (zeptomoles); nm (nanometers); kdal (kilodaltons);OD (optical density); EDTA (ethylene diamine tetra-acetic acid); FITC(fluorescein isothiocyanate); SDS (sodium dodecyl sulfate); NaPO₄(sodium phosphate); NP-40 (Nonidet P-40); Tris(tris(hydroxymethyl)-aminomethane); PMSF (phenylmethylsulfonylfluoride);TBE (Tris-Borate-EDTA, i.e., Tris buffer titrated with boric acid ratherthan HCl and containing EDTA); PBS (phosphate buffered saline); PPBS(phosphate buffered saline containing 1 mM PMSF); PAGE (polyacrylamidegel electrophoresis); Tween (polyoxyethylene-sorbitan); ATCC (AmericanType Culture Collection, Rockville, Md.); Coriell (Coriell CellRepositories, Camden, N.J.); DSMZ (Deutsche Sammlung von Mikroorganismenund Zellculturen, Braunschweig, Germany); Ambion (Ambion, Inc., Austin,Tex.); Boehringer (Boehringer Mannheim Biochemical, Indianapolis, Ind.);MJ Research (MJ Research, Watertown, Mass.; Sigma (Sigma ChemicalCompany, St. Louis, Mo.); Dynal (Dynal A. S., Oslo, Norway); Gull (GullLaboratories, Salt Lake City, Utah); Epicentre (Epicentre Technologies,Madison, Wis.); Lampire (Biological Labs., Inc., Coopersberg, Pa.); MJResearch (MJ Research, Watertown, Mass.); National Biosciences (NationalBiosciences, Plymouth, Minn.); NEB (New England Biolabs, Beverly,Mass.); Novagen (Novagen, Inc., Madison, Wis.); Perkin Elmer(Perkin-Elmer/ABI, Norwalk, Conn.); Promega (Promega, Corp., Madison,Wis.); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.);Clonetech (Clonetech, Palo Alto, Calif.) Pharmacia (Pharmacia,Piscataway, N.J.); Milton Roy (Milton Roy, Rochester, N.Y.); Amersham(Amersham International, Chicago, Ill.); and USB (U.S. Biochemical,Cleveland, Ohio). Glen Research (Glen Research, Sterling, Va.); Coriell(Coriell Cell Repositories, Camden, N.J.); Gentra (Gentra, Minneapolis,Minn.); Third Wave Technologies (Third Wave Technologies, Madison,Wis.); PerSeptive Biosystems (PerSeptive Biosystems, Framington, Mass.);Microsoft (Microsoft, Redmond, Wash.); Qiagen (Qiagen, Valencia,Calif.); Molecular Probes (Molecular Probes, Eugene, Oreg.); VWR (VWRScientific, ); Advanced Biotechnologies (Advanced Biotechnologies, INC.,Columbia, Md.); and Perkin elmer (Perkin Elmer,).

Example 1

[0607] Characteristics of Native Thermostable DNA Polymerases

[0608] A. 5′ Nuclease Activity of DNAPTaq

[0609] During the polymerase chain reaction (PCR) (Saiki et al., Science239:487 [1988]; Mullis and Faloona, Meth. Enzymol., 155:335 [1987]),DNAPTaq is able to amplify many, but not all, DNA sequences. Onesequence that cannot be amplified using DNAPTaq is shown in FIG. 5(Hairpin structure is SEQ ID NO: 15, FIG. 5 also shows a primer: SEQ IDNO: 17) This DNA sequence has the distinguishing characteristic of beingable to fold on itself to form a hairpin with two single-stranded arms,which correspond to the primers used in PCR.

[0610] To test whether this failure to amplify is due to the 5′ nucleaseactivity of the enzyme, the abilities of DNAPTaq and DNAPStf to amplifythis DNA sequence during 30 cycles of PCR were compared. Syntheticoligonucleotides were obtained from The Biotechnology Center at theUniversity of Wisconsin-Madison. The DNAPTaq and DNAPStf were fromPerkin Elmer (i.e., AMPLITAQ DNA polymerase and the Stoffel fragment ofAMPLITAQ DNA polymerase). The substrate DNA comprised the hairpinstructure shown in FIG. 6 cloned in a double-stranded form into pUC19.The primers used in the amplification are listed as SEQ ID NOS: 16-17.Primer SEQ ID NO: 17 is shown annealed to the 3′ arm of the hairpinstructure in FIG. 5. Primer SEQ ID NO: 16 is shown as the first 20nucleotides in bold on the 5′ arm of the hairpin in FIG. 5.

[0611] Polymerase chain reactions comprised 1 ng of supercoiled plasmidtarget DNA, 5 pmoles of each primer, 40 μM each dNTP, and 2.5 units ofDNAPTaq or DNAPStf, in a 50 μl solution of 10 mM Tris.Cl pH 8.3. TheDNAPTaq reactions included 50 mM KCl and 1.5 mM MgCl₂. The temperatureprofile was 95° C. for 30 sec., 55° C. for 1 min. and 72° C. for 1 min.,through 30 cycles. Ten percent of each reaction was analyzed by gelelectrophoresis through 6% polyacrylamide (cross-linked 29:1) in abuffer of 45 mM Tris.Borate, pH 8.3, 1.4 mM EDTA.

[0612] The results are shown in FIG. 6. The expected product was made byDNAPStf (indicated simply as “S”) but not by DNAPTaq (indicated as “T”).It was concluded that the 5′ nuclease activity of DNAPTaq is responsiblefor the lack of amplification of this DNA sequence.

[0613] To test whether the 5′ unpaired nucleotides in the substrateregion of this structured DNA are removed by DNAPTaq, the fate of theend-labeled 5′ arm during four cycles of PCR was compared using the sametwo polymerases (FIG. 7). The hairpin templates, such as the onedescribed in FIG. 5, were made using DNAPStf and a ³²P-5′-end-labeledprimer. The 5′-end of the DNA was released as a few large fragments byDNAPTaq but not by DNAPStf. The sizes of these fragments (based on theirmobilities) show that they contain most or all of the unpaired 5′ arm ofthe DNA. Thus, cleavage occurs at or near the base of the bifurcatedduplex. These released fragments terminate with 3′OH groups, asevidenced by direct sequence analysis, and the abilities of thefragments to be extended by terminal deoxynucleotidyl transferase.

[0614] FIGS. 8-10 show the results of experiments designed tocharacterize the cleavage reaction catalyzed by DNAPTaq. Unlessotherwise specified, the cleavage reactions comprised 0.01 pmoles ofheat-denatured, end-labeled hairpin DNA (with the unlabeledcomplementary strand also present), 1 pmole primer (complementary to the3′ arm) and 0.5 units of DNAPTaq (estimated to be 0.026 pmoles) in atotal volume of 10 μl of 10 mM Tris-Cl, ph 8.5, 50 mM KCl and 1.5 mMMgCl₂. As indicated, some reactions had different concentrations of KCl,and the precise times and temperatures used in each experiment areindicated in the individual Figures. The reactions that included aprimer used the one shown in FIG. 5 (SEQ ID NO: 17). In some instances,the primer was extended to the junction site by providing polymerase andselected nucleotides.

[0615] Reactions were initiated at the final reaction temperature by theaddition of either the MgCl₂ or enzyme. Reactions were stopped at theirincubation temperatures by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. The T_(m) calculations listed were madeusing the Oligo™ primer analysis software from National Biosciences,Inc. These were determined using 0.25 μM as the DNA concentration, ateither 15 or 65 mM total salt (the 1.5 mM MgCl₂ in all reactions wasgiven the value of 15 mM salt for these calculations).

[0616]FIG. 8 is an autoradiogram containing the results of a set ofexperiments and conditions on the cleavage site. FIG. 8A is adetermination of reaction components that enable cleavage. Incubation of5′-end-labeled hairpin DNA was for 30 minutes at 55° C., with theindicated components. The products were resolved by denaturingpolyacrylamide gel electrophoresis and the lengths of the products, innucleotides, are indicated. FIG. 8B describes the effect of temperatureon the site of cleavage in the absence of added primer. Reactions wereincubated in the absence of KCl for 10 minutes at the indicatedtemperatures. The lengths of the products, in nucleotides, areindicated.

[0617] Surprisingly, cleavage by DNAPTaq requires neither a primer nordNTPs (See FIG. 8A). Thus, the 5′ nuclease activity can be uncoupledfrom polymerization. Nuclease activity requires magnesium ions, thoughmanganese ions can be substituted, albeit with potential changes inspecificity and activity. Neither zinc nor calcium ions support thecleavage reaction. The reaction occurs over a broad temperature range,from 25° C. to 85° C., with the rate of cleavage increasing at highertemperatures.

[0618] Still referring to FIG. 8, the primer is not elongated in theabsence of added dNTPs. However, the primer influences both the site andthe rate of cleavage of the hairpin. The change in the site of cleavage(FIG. 8A) apparently results from disruption of a short duplex formedbetween the arms of the DNA substrate. In the absence of primer, thesequences indicated by underlining in FIG. 5 could pair, forming anextended duplex. Cleavage at the end of the extended duplex wouldrelease the 11 nucleotide fragment seen on the FIG. 8A lanes with noadded primer. Addition of excess primer (FIG. 8A, lanes 3 and 4) orincubation at an elevated temperature (FIG. 8B) disrupts the shortextension of the duplex and results in a longer 5′ arm and, hence,longer cleavage products.

[0619] The location of the 3′ end of the primer can influence theprecise site of cleavage. Electrophoretic analysis revealed that in theabsence of primer (FIG. 8B), cleavage occurs at the end of the substrateduplex (either the extended or shortened form, depending on thetemperature) between the first and second base pairs. When the primerextends up to the base of the duplex, cleavage also occurs onenucleotide into the duplex. However, when a gap of four or sixnucleotides exists between the 3′ end of the primer and the substrateduplex, the cleavage site is shifted four to six nucleotides in the 5′direction.

[0620]FIG. 9 describes the kinetics of cleavage in the presence (FIG.9A) or absence (FIG. 9B) of a primer oligonucleotide. The reactions wererun at 55° C. with either 50 mM KCl (FIG. 9A) or 20 mM KCl (FIG. 9B).The reaction products were resolved by denaturing polyacrylamide gelelectrophoresis and the lengths of the products, in nucleotides, areindicated. “M”, indicating a marker, is a 5′ end-labeled 19-ntoligonucleotide. Under these salt conditions, FIGS. 9A and 9B indicatethat the reaction appears to be about twenty times faster in thepresence of primer than in the absence of primer. This effect on theefficiency may be attributable to proper alignment and stabilization ofthe enzyme on the substrate.

[0621] The relative influence of primer on cleavage rates becomes muchgreater when both reactions are run in 50 mM KCl. In the presence ofprimer, the rate of cleavage increases with KCl concentration, up toabout 50 mM. However, inhibition of this reaction in the presence ofprimer is apparent at 100 mM and is complete at 150 mM KCl. In contrast,in the absence of primer the rate is enhanced by concentration of KCl upto 20 mM, but it is reduced at concentrations above 30 mM. At 50 mM KCl,the reaction is almost completely inhibited. The inhibition of cleavageby KCl in the absence of primer is affected by temperature, being morepronounced at lower temperatures.

[0622] Recognition of the 5′ end of the arm to be cut appears to be animportant feature of substrate recognition. Substrates that lack a free5′ end, such as circular M13 DNA, cannot be cleaved under any conditionstested. Even with substrates having defined 5′ arms, the rate ofcleavage by DNAPTaq is influenced by the length of the arm. In thepresence of primer and 50 mM KCl, cleavage of a 5′ extension that is 27nucleotides long is essentially complete within 2 minutes at 55° C. Incontrast, cleavages of molecules with 5′ arms of 84 and 188 nucleotidesare only about 90% and 40% complete after 20 minutes. Incubation athigher temperatures reduces the inhibitory effects of long extensionsindicating that secondary structure in the 5′ arm or a heat-labilestructure in the enzyme may inhibit the reaction. A mixing experiment,run under conditions of substrate excess, shows that the molecules withlong arms do not preferentially tie up the available enzyme innon-productive complexes. These results may indicate that the 5′nuclease domain gains access to the cleavage site at the end of thebifurcated duplex by moving down the 5′ arm from one end to the other.Longer 5′ arms would be expected to have more adventitious secondarystructures (particularly when KCl concentrations are high), which wouldbe likely to impede this movement.

[0623] Cleavage does not appear to be inhibited by long 3′ arms ofeither the substrate strand target molecule or pilot nucleic acid, atleast up to 2 kilobases. At the other extreme, 3′ arms of the pilotnucleic acid as short as one nucleotide can support cleavage in aprimer-independent reaction, albeit inefficiently. Fully pairedoligonucleotides do not elicit cleavage of DNA templates during primerextension.

[0624] The ability of DNAPTaq to cleave molecules even when thecomplementary strand contains only one unpaired 3′ nucleotide may beuseful in optimizing allele-specific PCR. PCR primers that have unpaired3′ ends could act as pilot oligonucleotides to direct selective cleavageof unwanted templates during preincubation of potential template-primercomplexes with DNAPTaq in the absence of nucleoside triphosphates.

[0625] B. 5′ Nuclease Activities of Other DNAPs

[0626] To determine whether other 5′ nucleases in other DNAPs would besuitable for the present invention, an array of enzymes, several ofwhich were reported in the literature to be free of apparent 5′ nucleaseactivity, were examined. The ability of these other enzymes to cleavenucleic acids in a structure-specific manner was tested using thehairpin substrate shown in FIG. 5 under conditions reported to beoptimal for synthesis by each enzyme.

[0627] DNAPEcl and DNAP Klenow were obtained from Promega; the DNAP ofPyrococcus furious (“Pfu”, Bargseid et al., Strategies 4:34 [1991]) wasfrom Stratagene; the DNAP of Thermococcus litoralis (“Tli”, Vent™(exo-), Perler et al., Proc. Natl. Acad. Sci. USA 89:5577 [1992] wasfrom New England Biolabs; the DNAP of Thermus flavus (“Tfl”, Kaledin etal., Biokhimiya 46:1576 [1981] was from Epicentre Technologies; and theDNAP of Thermus thermophilus (“Tth”, Carballeira et al., Biotechn.,9:276 [1990]; Myers et al., Biochem., 30:7661 (1991)] was from U.S.Biochemicals. 0.5 units of each DNA polymerase was assayed in a 20 μlreaction, using either the buffers supplied by the manufacturers for theprimer-dependent reactions, or 10 mM Tris·Cl, pH 8.5, 1.5 mM MgCl₂, and20 mM KCl. Reaction mixtures were at held 72° C. before the addition ofenzyme.

[0628]FIG. 10 is an autoradiogram recording the results of these tests.FIG. 10A demonstrates reactions of endonucleases of DNAPs of severalthermophilic bacteria. The reactions were incubated at 55° C. for 10minutes in the presence of primer or at 72° C. for 30 minutes in theabsence of primer, and the products were resolved by denaturingpolyacrylamide gel electrophoresis. The lengths of the products, innucleotides, are indicated. FIG. 10B demonstrates endonucleolyticcleavage by the 5′ nuclease of DNAPEcl. The DNAPEcl and DNAP Klenowreactions were incubated for 5 minutes at 37° C. Note the light band ofcleavage products of 25 and 11 nucleotides in the DNAPEcl lanes (made inthe presence and absence of primer, respectively). FIG. 8A alsodemonstrates DNAPTaq reactions in the presence (+) or absence (−) ofprimer. These reactions were run in 50 mM and 20 mM KCl, respectively,and were incubated at 55° C. for 10 minutes.

[0629] Referring to FIG. 10A, DNAPs from the cubacteria Thermusthermophilus and Thermus flavus cleave the substrate at the same placeas DNAPTaq, both in the presence and absence of primer. In contrast,DNAPs from the archaebacteria Pyrococcus furiosus and Thermococcuslitoralis are unable to cleave the substrates endonucleolytically. TheDNAPs from Pyrococcus furious and Thermococcus litoralis share littlesequence homology with eubacterial enzymes (Ito et al., Nucl. Acids Res.19:4045 (1991); Mathur et al., Nucl. Acids. Res. 19:6952 (1991); seealso Perler et al.). Referring to FIG. 10B, DNAPEcl also cleaves thesubstrate, but the resulting cleavage products are difficult to detectunless the 3′ exonuclease is inhibited. The amino acid sequences of the5′ nuclease domains of DNAPEcl and DNAPTaq are about 38% homologous(Gelfand, supra).

[0630] The 5′ nuclease domain of DNAPTaq also shares about 19% homologywith the 5′ exonuclease encoded by gene 6 of bacteriophage T7 (Dunn etal., J. Mol. Biol., 166:477 [1983]). This nuclease, which is notcovalently attached to a DNAP polymerization domain, is also able tocleave DNA endonucleolytically, at a site similar or identical to thesite that is cut by the 5′ nucleases described above, in the absence ofadded primers.

[0631] C. Transcleavage

[0632] The ability of a 5′ nuclease to be directed to cleave efficientlyat any specific sequence was demonstrated in the following experiment. Apartially complementary oligonucleotide termed a “pilot oligonucleotide”was hybridized to sequences at the desired point of cleavage. Thenon-complementary part of the pilot oligonucleotide provided a structureanalogous to the 3′ arm of the template (see FIG. 5), whereas the 5′region of the substrate strand became the 5′ arm. A primer was providedby designing the 3′ region of the pilot so that it would fold on itselfcreating a short hairpin with a stabilizing tetra-loop (Antao et al.,Nucl. Acids Res. 19:5901 [1991 ). Two pilot oligonucleotides are shownin FIG. 11A. Oligonucleotides 19-12 (SEQ ID NO: 18), 30-12 (SEQ ID NO:19) and 30-0 (SEQ ID NO: 20) are 31, 42 or 30 nucleotides long,respectively. However, oligonucleotides 19-12 (SEQ ID NO: 18) and 34-19(SEQ ID NO: 19) have only 19 and 30 nucleotides, respectively, that arecomplementary to different sequences in the substrate strand. The pilotoligonucleotides are calculated to melt off their complements at about50° C. (19-12) and about 75° C. (30-12). Both pilots have 12 nucleotidesat their 3′ ends, which act as 3′ arms with base-paired primersattached.

[0633] To demonstrate that cleavage could be directed by a pilotoligonucleotide, a single-stranded target DNA with DNAPTaq was incubatedin the presence of two potential pilot oligonucleotides. Thetranscleavage reactions, where the target and pilot nucleic acids arenot covalently linked, includes 0.01 pmoles of single end-labeledsubstrate DNA, 1 unit of DNAPTaq and 5 pmoles of pilot oligonucleotidein a volume of 20 μl of the same buffers. These components were combinedduring a one minute incubation at 95° C., to denature the PCR-generateddouble-stranded substrate DNA, and the temperatures of the reactionswere then reduced to their final incubation temperatures.Oligonucleotides 30-12 and 19-12 can hybridize to regions of thesubstrate DNAs that are 85 and 27 nucleotides from the 5′ end of thetargeted strand.

[0634]FIG. 19 shows the complete 206-mer sequence (SEQ ID NO: 27). The206-mer was generated by PCR. The M13/pUC 24-mer reverse sequencing(−48) primer and the M13/pUC sequencing (−47) primer from NEB (cataloguenos. 1233 and 1224 respectively) were used (50 pmoles each) with thepGEM3z(f+) plasmid vector (Promega) as template (10 ng) containing thetarget sequences. The conditions for PCR were as follows: 50 μM of eachdNTP and 2.5 units of Taq DNA polymerase in 100 μl of 20 mM Tris-Cl, pH8.3, 1.5 mM MgCl₂, 50 mM KCl with 0.05% Tween-20 and 0.05% NP-40.Reactions were cycled 35 times through 95° C. for 45 seconds, 63° C. for45 seconds, then 72° C. for 75 seconds. After cycling, reactions werefinished off with an incubation at 72° C. for 5 minutes. The resultingfragment was purified by electrophoresis through a 6% polyacrylamide gel(29:1 cross link) in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA,visualized by ethidium bromide staining or autoradiography, excised fromthe gel, eluted by passive diffusion, and concentrated by ethanolprecipitation.

[0635] Cleavage of the substrate DNA occurred in the presence of thepilot oligonucleotide 19-12 at 50° C. (FIG. 11B, lanes 1 and 7) but notat 75° C. (lanes 4 and 10). In the presence of oligonucleotide 30-12cleavage was observed at both temperatures. Cleavage did not occur inthe absence of added oligonucleotides (lanes 3, 6 and 12) or at about80° C. even though at 50° C. adventitious structures in the substrateallowed primer-independent cleavage in the absence of KCl (FIG. 11B,lane 9). A non-specific oligonucleotide with no complementarity to thesubstrate DNA did not direct cleavage at 50° C., either in the absenceor presence of 50 mM KCl (lanes 13 and 14). Thus, the specificity of thecleavage reactions can be controlled by the extent of complementarity tothe substrate and by the conditions of incubation.

[0636] D. Cleavage of RNA

[0637] A shortened RNA version of the sequence used in the transcleavageexperiments discussed above was tested for its ability to serve as asubstrate in the reaction. The RNA is cleaved at the expected place, ina reaction that is dependent upon the presence of the pilotoligonucleotide. The RNA substrate, made by T7 RNA polymerase in thepresence of (α-³²P)UTP, corresponds to a truncated version of the DNAsubstrate used in FIG. 11B. Reaction conditions were similar to those inused for the DNA substrates described above, with 50 mM KCl; incubationwas for 40 minutes at 55° C. The pilot oligonucleotide used is termed30-0 (SEQ ID NO: 20) and is shown in FIG. 12A.

[0638] The results of the cleavage reaction is shown in FIG. 13B. Thereaction was run either in the presence or absence of DNAPTaq or pilotoligonucleotide as indicated in FIG. 12B.

[0639] Strikingly, in the case of RNA cleavage, a 3′ arm is not requiredfor the pilot oligonucleotide. It is very unlikely that this cleavage isdue to previously described RNaseH, which would be expected to cut theRNA in several places along the 30 base-pair long RNA-DNA duplex. The 5′nuclease of DNAPTaq is a structure-specific RNaseH that cleaves the RNAat a single site near the 5′ end of the heteroduplexed region.

[0640] It is surprising that an oligonucleotide lacking a 3′ arm is ableto act as a pilot in directing efficient cleavage of an RNA targetbecause such oligonucleotides are unable to direct efficient cleavage ofDNA targets using native DNAPs. However, some 5′ nucleases of thepresent invention (for example, clones E, F and G of FIG. 4) can cleaveDNA in the absence of a 3′ arm. In other words, a non-extendablecleavage structure is not required for specific cleavage with some 5′nucleases of the present invention derived from thermostable DNApolymerases.

[0641] Tests were then conducted to determine whether cleavage of an RNAtemplate by DNAPTaq in the presence of a fully complementary primercould help explain why DNAPTaq is unable to extend a DNA oligonucleotideon an RNA template, in a reaction resembling that of reversetranscriptase. Another thermophilic DNAP, DNAPTth, is able to use RNA asa template, but only in the presence of Mn++, so it was predicted thatthis enzyme would not cleave RNA in the presence of this cation.Accordingly, an RNA molecule was incubated with an appropriate pilotoligonucleotide in the presence of DNAPTaq or DNAPTth, in buffercontaining either Mg++ or Mn++. As expected, both enzymes cleaved theRNA in the presence of Mg++. However, DNAPTaq, but not DNAPTth, degradedthe RNA in the presence of Mn++. It was concluded that the 5′ nucleaseactivities of many DNAPs may contribute to their inability to use RNA astemplates.

Example 2

[0642] Generation of 5′ Nucleases From Thermostable DNA Polymerases

[0643] Thermostable DNA polymerases were generated which have reducedsynthetic activity, an activity that is an undesirable side-reactionduring DNA cleavage in the detection assay of the invention, yet havemaintained thermostable nuclease activity. The result is a thermostablepolymerase which cleaves nucleic acids DNA with extreme specificity.

[0644] Type A DNA polymerases from eubacteria of the genus Thermus shareextensive protein sequence identity (90% in the polymerization domain,using the Lipman-Pearson method in the DNA analysis software fromDNAStar, Wis.) and behave similarly in both polymerization and nucleaseassays. Therefore, the genes for the DNA polymerase of Thermus aquaticus(DNAPTaq) and Thermus flavus (DNAPTfl) are used as representatives ofthis class. Polymerase genes from other eubacterial organisms, such asThermus thermophilus, Thermus sp., Thermotoga maritima, Thermosiphoafricanus and Bacillus stearothermophilus are equally suitable. The DNApolymerases from these thermophilic organisms are capable of survivingand performing at elevated temperatures, and can thus be used inreactions in which temperature is used as a selection againstnon-specific hybridization of nucleic acid strands.

[0645] The restriction sites used for deletion mutagenesis, describedbelow, were chosen for convenience. Different sites situated withsimilar convenience are available in the Thermus thermophilus gene andcan be used to make similar constructs with other Type A polymerasegenes from related organisms.

[0646] A. Creation of 5′ Nuclease Constructs

[0647] 1. Modified DNAPTaq Genes

[0648] The first step was to place a modified gene for the Taq DNApolymerase on a plasmid under control of an inducible promoter. Themodified Taq polymerase gene was isolated as follows: The Taq DNApolymerase gene was amplified by polymerase chain reaction from genomicDNA from Thermus aquaticus, strain YT-1 (Lawyer et al., supra), using asprimers the oligonucleotides described in SEQ ID NOS: 13-14. Theresulting fragment of DNA has a recognition sequence for the restrictionendonuclease EcoRI at the 5′ end of the coding sequence and a BglIIsequence at the 3′ end. Cleavage with BglII leaves a 5′ overhang or“sticky end” that is compatible with the end generated by BamHI. ThePCR-amplified DNA was digested with EcoRI and BamHI. The 2512 bpfragment containing the coding region for the polymerase gene was gelpurified and then ligated into a plasmid which contains an induciblepromoter.

[0649] In one embodiment of the invention, the pTTQ 18 vector, whichcontains the hybrid trp-lac (tac) promoter, was used (Stark, Gene 5:255[1987]) and shown in FIG. 13. The tac promoter is under the control ofthe E. coli lac repressor. Repression allows the synthesis of the geneproduct to be suppressed until the desired level of bacterial growth hasbeen achieved, at which point repression is removed by addition of aspecific inducer, isopropyl-β-D-thiogalactopyranoside (IPTG). Such asystem allows the expression of foreign proteins that may slow orprevent growth of transformants.

[0650] Bacterial promoters, such as tac, may not be adequatelysuppressed when they are present on a multiple copy plasmid. If a highlytoxic protein is placed under control of such a promoter, the smallamount of expression leaking through can be harmful to the bacteria. Inanother embodiment of the invention, another option for repressingsynthesis of a cloned gene product was used. The non-bacterial promoter,from bacteriophage T7, found in the plasmid vector series pET-3 was usedto express the cloned mutant Taq polymerase genes (FIG. 15; Studier andMoffatt, J. Mol. Biol., 189:113 [1986]). This promoter initiatestranscription only by T7 RNA polymerase. In a suitable strain, such asBL21(DE3)pLYS, the gene for this RNA polymerase is carried on thebacterial genome under control of the lac operator. This arrangement hasthe advantage that expression of the multiple copy gene (on the plasmid)is completely dependent on the expression of T7 RNA polymerase, which iseasily suppressed because it is present in a single copy.

[0651] For ligation into the pTTQ18 vector (FIG. 13), the PCR productDNA containing the Taq polymerase coding region (mutTaq, clone 4B, SEQID NO: 21) was digested with EcoRI and BglII and this fragment wasligated under standard “sticky end” conditions (Sambrook et al.Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, pp. 1.63-1.69 [1989]) into the EcoRI and BamHI sites of theplasmid vector pTTQ 18. Expression of this construct yields atranslational fusion product in which the first two residues of thenative protein (Met-Arg) are replaced by three from the vector(Met-Asn-Ser), but the remainder of the natural protein would notchange. The construct was transformed into the JM109 strain of E. coliand the transformants were plated under incompletely repressingconditions that do not permit growth of bacteria expressing the nativeprotein. These plating conditions allow the isolation of genescontaining pre-existing mutations, such as those that result from theinfidelity of Taq polymerase during the amplification process.

[0652] Using this amplification/selection protocol, a clone (depicted inFIG. 3B) containing a mutated Taq polymerase gene (mutTaq, clone 3B) wasisolated. The mutant was first detected by its phenotype, in whichtemperature-stable 5′ nuclease activity in a crude cell extract wasnormal, but polymerization activity was almost absent (approximatelyless than 1% of wild type Taq polymerase activity).

[0653] DNA sequence analysis of the recombinant gene showed that it hadchanges in the polymerase domain resulting in two amino acidsubstitutions: an A to G change at nucleotide position 1394 causes a Gluto Gly change at amino acid position 465 (numbered according to thenatural nucleic and amino acid sequences, SEQ ID NOS: 1 and 4) andanother A to G change at nucleotide position 2260 causes a Gln to Argchange at amino acid position 754. Because the Gln to Gly mutation is ata nonconserved position and because the Glu to Arg mutation alters anamino acid that is conserved in virtually all of the known Type Apolymerases, this latter mutation is most likely the one responsible forcurtailing the synthesis activity of this protein. The nucleotidesequence for the FIG. 3B construct is given in SEQ ID NO: 21. The enzymeencoded by this sequence is referred to as Cleavase® A/G.

[0654] Subsequent derivatives of DNAPTaq constructs were made from themutTaq gene, thus, they all bear these amino acid substitutions inaddition to their other alterations, unless these particular regionswere deleted. These mutated sites are indicated by black boxes at theselocations in the diagrams in FIG. 3. In FIG. 3, the designation “3′ Exo”is used to indicate the location of the 3′ exonuclease activityassociated with Type A polymerases which is not present in DNAPTaq. Allconstructs except the genes shown in FIGS. 3E, F and G were made in thepTTQ18 vector.

[0655] The cloning vector used for the genes in FIGS. 3E and F was fromthe commercially available pET-3 series, described above. Though thisvector series has only a BamHI site for cloning downstream of the T7promoter, the series contains variants that allow cloning into any ofthe three reading frames. For cloning of the PCR product describedabove, the variant called pET-3c was used (FIG. 14). The vector wasdigested with BamHI, dephosphorylated with calf intestinal phosphatase,and the sticky ends were filled in using the Klenow fragment of DNAPEcland dNTPs. The gene for the mutant Taq DNAP shown in FIG. 3B (mutTaq,clone 3B) was released from pTTQ18 by digestion with EcoRI and SalI, andthe “sticky ends” were filled in as was done with the vector. Thefragment was ligated to the vector under standard blunt-end conditions(Sambrook et al., Molecular Cloning, supra), the construct wastransformed into the BL21 (DE3)pLYS strain of E. coli, and isolates werescreened to identify those that were ligated with the gene in the properorientation relative to the promoter. This construction yields anothertranslational fusion product, in which the first two amino acids ofDNAPTaq (Met-Arg) are replaced by 13 from the vector plus two from thePCR primer (Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg-Ile-Asn-Ser)(SEQ ID NO: 24).

[0656] In these experiments, the goal was to generate enzymes thatlacked the ability to synthesize DNA, but retained the ability to cleavenucleic acids with a 5′ nuclease activity. The act of primed, templatedsynthesis of DNA is actually a coordinated series of events, so it ispossible to disable DNA synthesis by disrupting one event while notaffecting the others. These steps include, but are not limited to,primer recognition and binding, dNTP binding and catalysis of theinter-nucleotide phosphodiester bond. Some of the amino acids in thepolymerization domain of DNAPEcI have been linked to these functions,but the precise mechanisms are as yet poorly defined.

[0657] One way of destroying the polymerizing ability of a DNApolymerase is to delete all or part of the gene segment that encodesthat domain for the protein, or to otherwise render the gene incapableof making a complete polymerization domain. Individual mutant enzymesmay differ from each other in stability and solubility both inside andoutside cells. For instance, in contrast to the 5′ nuclease domain ofDNAPEcI, which can be released in an active form from the polymerizationdomain by gentle proteolysis (Setlow and Komberg, J. Biol. Chem.,247:232 [1972]), the Thermus nuclease domain, when treated similarly,becomes less soluble and the cleavage activity is often lost.

[0658] Using the mutant gene shown in FIG. 3B as starting material,several deletion constructs were created. All cloning technologies werestandard (Sambrook et al., supra) and are summarized briefly, asfollows:

[0659]FIG. 3C: The mutTaq construct was digested with PstI, which cutsonce within the polymerase coding region, as indicated, and cutsimmediately downstream of the gene in the multiple cloning site of thevector. After release of the fragment between these two sites, thevector was re-ligated, creating an 894-nucleotide deletion, and bringinginto frame a stop codon 40 nucleotides downstream of the junction. Thenucleotide sequence of this 5′ nuclease (clone 4C) is given in SEQ IDNO: 9.

[0660]FIG. 3D: The mutTaq construct was digested with NheI, which cutsonce in the gene at position 2047. The resulting four-nucleotide 5′overhanging ends were filled in, as described above, and the blunt endswere re-ligated. The resulting four-nucleotide insertion changes thereading frame and causes termination of translation ten amino acidsdownstream of the mutation. The nucleotide sequence of this 5′ nuclease(clone 3D) is given in SEQ ID NO: 10.

[0661]FIG. 3E: The entire mutTaq gene was cut from pTTQ18 using EcoRIand SalI and cloned into pET-3c, as described above. This clone wasdigested with BstXI and XcmI, at unique sites that are situated as shownin FIG. 3E. The DNA was treated with the Klenow fragment of DNAPEcl anddNTPs, which resulted in the 3′ overhangs of both sites being trimmed toblunt ends. These blunt ends were ligated together, resulting in anout-of-frame deletion of 1540 nucleotides. An in-frame termination codonoccurs 18 triplets past the junction site. The nucleotide sequence ofthis 5′ nuclease (clone 3E) is given in SEQ ID NO: 11, with theappropriate leader sequence given in SEQ ID NO: 25. It is also referredto as Cleavase® BX.

[0662]FIG. 3F: The entire mutTaq gene was cut from pTTQ18 using EcoRIand SalI and cloned into pET-3c, as described above. This clone wasdigested with BstXI and BamHI, at unique sites that are situated asshown in the diagram. The DNA was treated with the Klenow fragment ofDNAPEcl and dNTPs, which resulted in the 3′ overhang of the BstXI sitebeing trimmed to a blunt end, while the 5′ overhang of the BamHI sitewas filled in to make a blunt end. These ends were ligated together,resulting in an in-frame deletion of 903 nucleotides. The nucleotidesequence of the 5′ nuclease (clone 3F) is given in SEQ ID NO: 12. It isalso referred to as Cleavase® BB.

[0663]FIG. 3G: This polymerase is a variant of that shown in FIG. 4E. Itwas cloned in the plasmid vector pET-21 (Novagen). The non-bacterialpromoter from bacteriophage T7, found in this vector, initiatestranscription only by T7 RNA polymerase. See Studier and Moffatt, supra.In a suitable strain, such as (DES)pLYS, the gene for this RNApolymerase is carried on the bacterial genome under control of the lacoperator. This arrangement has the advantage that expression of themultiple copy gene (on the plasmid) is completely dependent on theexpression of T7 RNA polymerase, which is easily suppressed because itis present in a single copy. Because the expression of these mutantgenes is under this tightly controlled promoter, potential problems oftoxicity of the expressed proteins to the host cells are less of aconcern.

[0664] The pET-21 vector also features a “His*Tag”, a stretch of sixconsecutive histidine residues that are added on the carboxy terminus ofthe expressed proteins. The resulting proteins can then be purified in asingle step by metal chelation chromatography, using a commerciallyavailable (Novagen) column resin with immobilized Ni⁺⁺ ions. The 2.5 mlcolumns are reusable, and can bind up to 20 mg of the target proteinunder native or denaturing (guanidine*HCl or urea) conditions.

[0665]E. coli (DES)pLYS cells are transformed with the constructsdescribed above using standard transformation techniques, and used toinoculate a standard growth medium (e.g., Luria-Bertani broth).Production of T7 RNA polymerase is induced during log phase growth byaddition of IPTG and incubated for a further 12 to 17 hours. Aliquots ofculture are removed both before and after induction and the proteins areexamined by SDS-PAGE. Staining with Coomassie Blue allows visualizationof the foreign proteins if they account for about 3-5% of the cellularprotein and do not co-migrate with any of the major protein bands.Proteins that co-migrate with major host protein must be expressed asmore than 10% of the total protein to be seen at this stage of analysis.

[0666] Some mutant proteins are sequestered by the cells into inclusionbodies. These are granules that form in the cytoplasm when bacteria aremade to express high levels of a foreign protein, and they can bepurified from a crude lysate, and analyzed by SDS-PAGE to determinetheir protein content. If the cloned protein is found in the inclusionbodies, it must be released to assay the cleavage and polymeraseactivities. Different methods of solubilization may be appropriate fordifferent proteins, and a variety of methods are known (See e.g.,Builder & Ogez, U.S. Pat. No. 4,511,502 (1985); Olson, U.S. Pat. No.4,518,526 (1985); Olson & Pai, U.S. Pat. No. 4,511,503 (1985); and Joneset al., U.S. Pat. No. 4,512,922 (1985), all of which are herebyincorporated by reference).

[0667] The solubilized protein is then purified on the Ni⁺⁺ column asdescribed above, following the manufacturers instructions (Novagen). Thewashed proteins are eluted from the column by a combination of imidazolecompetitor (1 M) and high salt (0.5 M NaCl), and dialyzed to exchangethe buffer and to allow denature proteins to refold. Typical recoveriesresult in approximately 20 μg of specific protein per ml of startingculture. The DNAP mutant is referred to as the CLEAVASE BN nuclease andthe sequence is given in SEQ ID NO: 26 (the amino acid sequence of theCLEAVASE BN nuclease is obtained by translating the DNA sequence of SEQID NO: 26).

[0668] 2. Modified DNAPTfl Gene

[0669] The DNA polymerase gene of Thermus flavus was isolated from the“T. flavus” AT-62 strain obtained from the American Type TissueCollection (ATCC 33923). This strain has a different restriction mapthen does the T. flavus strain used to generate the sequence publishedby Akhmetzjanov and Vakhitov, supra. The published sequence is listed asSEQ ID NO: 2. No sequence data has been published for the DNA polymerasegene from the AT-62 strain of T. flavus.

[0670] Genomic DNA from T. flavus was amplified using the same primersused to amplify the T. aquaticus DNA polymerase gene (SEQ ID NOS:13-14). The approximately 2500 base pair PCR fragment was digested withEcoRI and BamHI. The over-hanging ends were made blunt with the Klenowfragment of DNAPEcl and dNTPs. The resulting approximately 1800 basepair fragment containing the coding region for the N-terminus wasligated into pET-3c, as described above. This construct, clone 4B, isdepicted in FIG. 4B. The wild type T. flavus DNA polymerase gene isdepicted in FIG. 4A. The 4B clone has the same leader amino acids as dothe DNAPTaq clones 4E and F which were cloned into pET-3c; it is notknown precisely where translation termination occurs, but the vector hasa strong transcription termination signal immediately downstream of thecloning site.

[0671] B. Growth and Induction of Transformed Cells

[0672] Bacterial cells were transformed with the constructs describedabove using standard transformation techniques and used to inoculate 2mls of a standard growth medium (e.g., Luria-Bertani broth). Theresulting cultures were incubated as appropriate for the particularstrain used, and induced if required for a particular expression system.For all of the constructs depicted in FIGS. 3 and 4, the cultures weregrown to an optical density (at 600 nm wavelength) of 0.5 OD.

[0673] To induce expression of the cloned genes, the cultures werebrought to a final concentration of 0.4 mM IPTG and the incubations werecontinued for 12 to 17 hours. Then, 50 μl aliquots of each culture wereremoved both before and after induction and were combined with 20 μl ofa standard gel loading buffer for sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE). Subsequent staining with Coomassie Blue(Sambrook et al., supra) allows visualization of the foreign proteins ifthey account for about 3-5% of the cellular protein and do notco-migrate with any of the major E. coli protein bands. Proteins that doco-migrate with a major host protein must be expressed as more than 10%of the total protein to be seen at this stage of analysis.

[0674] C. Heat Lysis and Fractionation

[0675] Expressed thermostable proteins (i.e., the 5′ nucleases), wereisolated by heating crude bacterial cell extracts to cause denaturationand precipitation of the less stable E. coli proteins. The precipitatedE. coli proteins were then, along with other cell debris, removed bycentrifugation. Then, 1.7 mls of the culture were pelleted bymicrocentrifugation at 12,000 to 14,000 rpm for 30 to 60 seconds. Afterremoval of the supernatant, the cells were resuspended in 400 μl ofbuffer A (50 mM Tris-HCl, pH 7.9, 50 mM dextrose, 1 mM EDTA),re-centrifuged, then resuspended in 80 μl of buffer A with 4 mg/mllysozyme. The cells were incubated at room temperature for 15 minutes,then combined with 80 μl of buffer B (10 mM Tris-HCl, pH 7.9, 50 mM KCl,1 mM EDTA, 1 mM PMSF, 0.5% Tween-20, 0.5% Nonidet-P40).

[0676] This mixture was incubated at 75° C. for 1 hour to denature andprecipitate the host proteins. This cell extract was centrifuged at14,000 rpm for 15 minutes at 4° C., and the supernatant was transferredto a fresh tube. An aliquot of 0.5 to 1 μl of this supernatant was useddirectly in each test reaction, and the protein content of the extractwas determined by subjecting 7 μl to electrophoretic analysis, as above.The native recombinant Taq DNA polymerase (Engelke, Anal. Biochem.,191:396 [1990]), and the double point mutation protein shown in FIG. 3Bare both soluble and active at this point.

[0677] The foreign protein may not be detected after the heat treatmentsdue to sequestration of the foreign protein by the cells into inclusionbodies. These are granules that form in the cytoplasm when bacteria aremade to express high levels of a foreign protein, and they can bepurified from a crude lysate, and analyzed SDS PAGE to determine theirprotein content. Many methods have been described in the literature, andone approach is described below.

[0678] D. Isolation and Solubilization of Inclusion Bodies

[0679] A small culture was grown and induced as described above. A 1.7ml aliquot was pelleted by brief centrifugation, and the bacterial cellswere resuspended in 100 μl of Lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mMEDTA, 100 mM NaCl). Then, 2.5 μl of 20 mM PMSF were added for a finalconcentration of 0.5 mM, and lysozyme was added to a concentration of1.0 mg/ml. The cells were incubated at room temperature for 20 minutes,deoxycholic acid was added to 1 mg/ml (1 μl of 100 mg/ml solution), andthe mixture was further incubated at 37° C. for about 15 minutes oruntil viscous. DNAse I was added to 10 μg/ml and the mixture wasincubated at room temperature for about 30 minutes or until it was nolonger viscous.

[0680] From this mixture the inclusion bodies were collected bycentrifugation at 14,000 rpm for 15 minutes at 4° C., and thesupernatant was discarded. The pellet was resuspended in 100 μl of lysisbuffer with 10 mM EDTA (pH 8.0) and 0.5% Triton X-100. After 5 minutesat room temperature, the inclusion bodies were pelleted as before, andthe supernatant was saved for later analysis. The inclusion bodies wereresuspended in 50 μl of distilled water, and 5 μl was combined with SDSgel loading buffer (which dissolves the inclusion bodies) and analyzedelectrophoretically, along with an aliquot of the supernatant.

[0681] If the cloned protein is found in the inclusion bodies, it may bereleased to assay the cleavage and polymerase activities and the methodof solubilization must be compatible with the particular activity.Different methods of solubilization may be appropriate for differentproteins, and a variety of methods are discussed in Molecular Cloning(Sambrook et al., supra). The following is an adaptation used forseveral of the isolates used in the development of the presentinvention.

[0682] Twenty μl of the inclusion body-water suspension were pelleted bycentrifugation at 14,000 rpm for 4 minutes at room temperature, and thesupernatant was discarded. To further wash the inclusion bodies, thepellet was resuspended in 20 μl of lysis buffer with 2M urea, andincubated at room temperature for one hour. The washed inclusion bodieswere then resuspended in 2 μl of lysis buffer with 8 M urea; thesolution clarified visibly as the inclusion bodies dissolved.Undissolved debris was removed by centrifugation at 14,000 rpm for 4minutes at room temperature, and the extract supernatant was transferredto a fresh tube.

[0683] To reduce the urea concentration, the extract was diluted intoKH₂PO₄. A fresh tube was prepared containing 180 μl of 50 mM KH₂PO₄, pH9.5, 1 mM EDTA and 50 mM NaCl. A 2 μl aliquot of the extract was addedand vortexed briefly to mix. This step was repeated until all of theextract had been added for a total of 10 additions. The mixture wasallowed to sit at room temperature for 15 minutes, during which timesome precipitate often forms. Precipitates were removed bycentrifugation at 14,000 rpm, for 15 minutes at room temperature, andthe supernatant was transferred to a fresh tube. To the 200 μl ofprotein in the KH₂PO₄ solution, 140-200 μl of saturated (NH₄)₂SO₄ wereadded, so that the resulting mixture was about 41% to 50% saturated(NH₄)₂SO₄. The mixture was chilled on ice for 30 minutes to allow theprotein to precipitate, and the protein was then collected bycentrifugation at 14,000 rpm, for 4 minutes at room temperature. Thesupernatant was discarded, and the pellet was dissolved in 20 μl BufferC (20 mM HEPES, pH 7.9, 1 mM EDTA, 0.5% PMSF, 25 mM KCl and 0.5 % eachof Tween-20 and Nonidet P 40). The protein solution was centrifugedagain for 4 minutes to pellet insoluble materials, and the supernatantwas removed to a fresh tube. The protein contents of extracts preparedin this manner were visualized by resolving 1-4 μl by SDS-PAGE; 0.5 to 1μl of extract was tested in the cleavage and polymerization assays asdescribed.

[0684] E. Protein Analysis for Presence of Nuclease and SyntheticActivity

[0685] The 5′ nucleases described above and shown in FIGS. 3 and 4 wereanalyzed by the following methods.

[0686] 1. Structure Specific Nuclease Assay

[0687] A candidate modified polymerase is tested for 5′ nucleaseactivity by examining its ability to catalyze structure-specificcleavages. By the term “cleavage structure” as used herein, is meant anucleic acid structure which is a substrate for cleavage by the 5′nuclease activity of a DNAP.

[0688] The polymerase is exposed to test complexes that have thestructures shown in FIG. 15. Testing for 5′ nuclease activity involvesthree reactions: 1) a primer-directed cleavage (FIG. 15B) is performedbecause it is relatively insensitive to variations in the saltconcentration of the reaction and can, therefore, be performed inwhatever solute conditions the modified enzyme requires for activity;this is generally the same conditions preferred by unmodifiedpolymerases; 2) a similar primer-directed cleavage is performed in abuffer which permits primer-independent cleavage (i.e., a low saltbuffer), to demonstrate that the enzyme is viable under theseconditions; and 3) a primer-independent cleavage (FIG. 15A) is performedin the same low salt buffer.

[0689] The bifurcated duplex is formed between a substrate strand and atemplate strand as shown in FIG. 15. By the term “substrate strand” asused herein, is meant that strand of nucleic acid in which the cleavagemediated by the 5′ nuclease activity occurs. The substrate strand isalways depicted as the top strand in the bifurcated complex which servesas a substrate for 5′ nuclease cleavage (FIG. 15). By the term “templatestrand” as used herein, is meant the strand of nucleic acid which is atleast partially complementary to the substrate strand and which annealsto the substrate strand to form the cleavage structure. The templatestrand is always depicted as the bottom strand of the bifurcatedcleavage structure (FIG. 15). If a primer (a short oligonucleotide of 19to 30 nucleotides in length) is added to the complex, as whenprimer-dependent cleavage is to be tested, it is designed to anneal tothe 3′ arm of the template strand (FIG. 15B). Such a primer would beextended along the template strand if the polymerase used in thereaction has synthetic activity.

[0690] The cleavage structure may be made as a single hairpin molecule,with the 3′ end of the target and the 5′ end of the pilot joined as aloop as shown in FIG. 15E. A primer oligonucleotide complementary to the3′ arm is also required for these tests so that the enzyme's sensitivityto the presence of a primer may be tested.

[0691] Nucleic acids to be used to form test cleavage structures can bechemically synthesized, or can be generated by standard recombinant DNAtechniques. By the latter method, the hairpin portion of the moleculecan be created by inserting into a cloning vector duplicate copies of ashort DNA segment, adjacent to each other but in opposing orientation.The double-stranded fragment encompassing this inverted repeat, andincluding enough flanking sequence to give short (about 20 nucleotides)unpaired 5′ and 3′ arms, can then be released from the vector byrestriction enzyme digestion, or by PCR performed with an enzyme lackinga 5′ exonuclease (e.g., the Stoffel fragment of AMPLITAQ DNA polymerase,Vent™ DNA polymerase).

[0692] The test DNA can be labeled on either end, or internally, witheither a radioisotope, or with a non-isotopic tag. Whether the hairpinDNA is a synthetic single strand or a cloned double strand, the DNA isheated prior to use to melt all duplexes. When cooled on ice, thestructure depicted in FIG. 16E is formed, and is stable for sufficienttime to perform these assays.

[0693] To test for primer-directed cleavage (Reaction 1), a detectablequantity of the test molecule (typically 1-100 fmol of ³²P-labeledhairpin molecule) and a 10 to 100-fold molar excess of primer are placedin a buffer known to be compatible with the test enzyme. For Reaction 2,where primer-directed cleavage is performed under condition which allowprimer-independent cleavage, the same quantities of molecules are placedin a solution that is the same as the buffer used in Reaction 1regarding pH, enzyme stabilizers (e.g., bovine serum albumin, nonionicdetergents, gelatin) and reducing agents (e.g., dithiothreitol,2-mercaptoethanol) but that replaces any monovalent cation salt with 20mM KCl; 20 mM KCl is the demonstrated optimum for primer-independentcleavage. Buffers for enzymes, such as DNAPEcl, that usually operate inthe absence of salt are not supplemented to achieve this concentration.To test for primer-independent cleavage (Reaction 3) the same quantityof the test molecule, but no primer, are combined under the same bufferconditions used for Reaction 2.

[0694] All three test reactions are then exposed to enough of the enzymethat the molar ratio of enzyme to test complex is approximately 1:1. Thereactions are incubated at a range of temperatures up to, but notexceeding, the temperature allowed by either the enzyme stability or thecomplex stability, whichever is lower, up to 80° C. for enzymes fromthermophiles, for a time sufficient to allow cleavage (10 to 60minutes). The products of Reactions 1, 2 and 3 are resolved bydenaturing polyacrylamide gel electrophoresis, and visualized byautoradiography or by a comparable method appropriate to the labelingsystem used. Additional labeling systems include chemiluminescencedetection, silver or other stains, blotting and probing and the like.The presence of cleavage products is indicated by the presence ofmolecules which migrate at a lower molecular weight than does theuncleaved test structure. These cleavage products indicate that thecandidate polymerase has structure-specific 5′ nuclease activity.

[0695] To determine whether a modified DNA polymerase has substantiallythe same 5′ nuclease activity as that of the native DNA polymerase, theresults of the above-described tests are compared with the resultsobtained from these tests performed with the native DNA polymerase. By“substantially the same 5′ nuclease activity” it is meant that themodified polymerase and the native polymerase will both cleave testmolecules in the same manner. It is not necessary that the modifiedpolymerase cleave at the same rate as the native DNA polymerase.

[0696] Some enzymes or enzyme preparations may have other associated orcontaminating activities that may be functional under the cleavageconditions described above and that may interfere with 5′ nucleasedetection. Reaction conditions can be modified in consideration of theseother activities, to avoid destruction of the substrate, or othermasking of the 5′ nuclease cleavage and its products. For example, theDNA polymerase I of E. coli (Pol I), in addition to its polymerase and5′ nuclease activities, has a 3′ exonuclease that can degrade DNA in a3′ to 5′ direction. Consequently, when the molecule in FIG. 15E isexposed to this polymerase under the conditions described above, the 3′exonuclease quickly removes the unpaired 3′ arm, destroying thebifurcated structure required of a substrate for the 5′ exonucleasecleavage and no cleavage is detected. The true ability of Pol I tocleave the structure can be revealed if the 3′ exonuclease is inhibitedby a change of conditions (e.g., pH), mutation, or by addition of acompetitor for the activity. Addition of 500 pmoles of a single-strandedcompetitor oligonucleotide, unrelated to the FIG. 15E structure, to thecleavage reaction with Pol I effectively inhibits the digestion of the3′ arm of the FIG. 15E structure without interfering with the 5′exonuclease release of the 5′ arm. The concentration of the competitoris not critical, but should be high enough to occupy the 3′ exonucleasefor the duration of the reaction.

[0697] Similar destruction of the test molecule may be caused bycontaminants in the candidate polymerase preparation. Several sets ofthe structure specific nuclease reactions may be performed to determinethe purity of the candidate nuclease and to find the window betweenunder and over exposure of the test molecule to the polymerasepreparation being investigated.

[0698] The above described modified polymerases were tested for 5′nuclease activity as follows: Reaction 1 was performed in a buffer of 10mM Tris-Cl, pH 8.5 at 20° C., 1.5 mM MgCl₂ and 50 mM KCl and in Reaction2 the KCl concentration was reduced to 20 mM. In Reactions 1 and 2, 10fmoles of the test substrate molecule shown in FIG. 15E were combinedwith 1 pmole of the indicated primer and 0.5 to 1.0 μl of extractcontaining the modified polymerase (prepared as described above). Thismixture was lo then incubated for 10 minutes at 55° C. For all of themutant polymerases tested these conditions were sufficient to givecomplete cleavage. When the molecule shown in FIG. 15E was labeled atthe 5′ end, the released 5′ fragment, 25 nucleotides long, wasconveniently resolved on a 20% polyacrylamide gel (19:1 cross-linked)with 7 M urea in a buffer containing 45 mM Tris-borate pH 8.3, 1.4 mMEDTA. Clones 3C-F and 4B exhibited structure-specific cleavagecomparable to that of the unmodified DNA polymerase. Additionally,clones 3E, 3F and 3G have the added ability to cleave DNA in the absenceof a 3′ arm as discussed above. Representative cleavage reactions areshown in FIG. 16.

[0699] For the reactions shown in FIG. 16, the mutant polymerase clones3E (Taq mutant) and 4B (Tfl mutant) were examined for their ability tocleave the hairpin substrate molecule shown in FIG. 15E. The substratemolecule was labeled at the 5′ terminus with ³²P. Ten fmoles ofheat-denatured, end-labeled substrate DNA and 0.5 units of DNAPTaq(lane 1) or 0.5 μl of 3E or 4B extract (FIG. 16, lanes 2-7, extract wasprepared as described above) were mixed together in a buffer containing10 mM Tris-Cl, pH 8.5, 50 mM KCl and 1.5 mM MgCl₂. The final reactionvolume was 10 μl. Reactions shown in lanes 4 and 7 contain in addition50 μM of each dNTP. Reactions shown in lanes 3, 4, 6 and 7 contain 0.2μM of the primer oligonucleotide (complementary to the 3′ arm of thesubstrate and shown in FIG. 15E). Reactions were incubated at 55° C. for4 minutes. Reactions were stopped by the addition of 8 μl of 95%formamide containing 20 mM EDTA and 0.05% marker dyes per 10 μl reactionvolume. Samples were then applied to 12% denaturing acrylamide gels.Following electrophoresis, the gels were autoradiographed. FIG. 16 showsthat clones 3E and 4B exhibit cleavage activity similar to that of thenative DNAPTaq. Note that some cleavage occurs in these reactions in theabsence of the primer. When long hairpin structure, such as the one usedhere (FIG. 15E), are used in cleavage reactions performed in bufferscontaining 50 mM KCl a low level of primer-independent cleavage is seen.Higher concentrations of KCl suppress, but do not eliminate, thisprimer-independent cleavage under these conditions.

[0700] 2. Assay for Synthetic Activity

[0701] The ability of the modified enzyme or proteolytic fragments isassayed by adding the modified enzyme to an assay system in which aprimer is annealed to a template and DNA synthesis is catalyzed by theadded enzyme. Many standard laboratory techniques employ such an assay.For example, nick translation and enzymatic sequencing involve extensionof a primer along a DNA template by a polymerase molecule.

[0702] In a preferred assay for determining the synthetic activity of amodified enzyme an oligonucleotide primer is annealed to asingle-stranded DNA template (e.g., bacteriophage M13 DNA), and theprimer/template duplex is incubated in the presence of the modifiedpolymerase in question, deoxynucleoside triphosphates (dNTPs) and thebuffer and salts known to be appropriate for the unmodified or nativeenzyme. Detection of either primer extension (by denaturing gelelectrophoresis) or dNTP incorporation (by acid precipitation orchromatography) is indicative of an active polymerase. A label, eitherisotopic or non-isotopic, is preferably included on either the primer oras a dNTP to facilitate detection of polymerization products. Syntheticactivity is quantified as the amount of free nucleotide incorporatedinto the growing DNA chain and is expressed as amount incorporated perunit of time under specific reaction conditions.

[0703] Representative results of an assay for synthetic activity isshown in FIG. 17. The synthetic activity of the mutant DNAPTaq clones3B-F was tested as follows: A master mixture of the following buffer wasmade: 1.2× PCR buffer (1× PCR buffer contains 50 mM KCl, 1.5 mM MgCl₂,10 mM Tris-Cl, pH 8.5 and 0.05% each Tween 20 and Nonidet P40), 50 μMeach of dGTP, dATP and dTTP, 5 μM dCTP and 0.125 μM α-³²P-dCTP at 600Ci/mmol. Before adjusting this mixture to its final volume, it wasdivided into two equal aliquots. One received distilled water up to avolume of 50 μl to give the concentrations above. The other received 5μg of single-stranded M13mp18 DNA (approximately 2.5 pmol or 0.05 μMfinal concentration) and 250 pmol of M13 sequencing primer (5 μM finalconcentration) and distilled water to a final volume of 50 μl. Eachcocktail was warmed to 75° C. for 5 minutes and then cooled to roomtemperature. This allowed the primers to anneal to the DNA in theDNA-containing mixtures.

[0704] For each assay, 4 μl of the cocktail with the DNA was combinedwith 1 μl of the mutant polymerase, prepared as described, or 1 unit ofDNAPTaq (Perkin Elmer) in 1 μl of dH₂O. A “no DNA” control was done inthe presence of the DNAPTaq (FIG. 17, lane 1), and a “no enzyme” controlwas done using water in place of the enzyme (lane 2). Each reaction wasmixed, then incubated at room temperature (approx. 22° C.) for 5minutes, then at 55° C. for 2 minutes, then at 72° C. for 2 minutes.This step incubation was done to detect polymerization in any mutantsthat might have optimal temperatures lower than 72° C. After the finalincubation, the tubes were spun briefly to collect any condensation andwere placed on ice. One ill of each reaction was spotted at an origin1.5 cm from the bottom edge of a polyethyleneimine (PEI) cellulose thinlayer chromatography plate and allowed to dry. The chromatography platewas run in 0.75 M NaH₂PO₄, pH 3.5, until the buffer front had runapproximately 9 cm from the origin. The plate was dried, wrapped inplastic wrap, marked with luminescent ink, and exposed to X-ray film.Incorporation was detected as counts that stuck where originallyspotted, while the unincorporated nucleotides were carried by the saltsolution from the origin.

[0705] Comparison of the locations of the counts with the two controllanes confirmed the lack of polymerization activity in the mutantpreparations. Among the modified DNAPTaq clones, only clone 3B retainsany residual synthetic activity as shown in FIG. 17.

Example 3

[0706] 5′ Nucleases Derived From Thermostable DNA Polymerases Can CleaveShort Hairpin Structures with Specificity

[0707] The ability of the 5′ nucleases to cleave hairpin structures togenerate a cleaved hairpin structure suitable as a detection moleculewas examined. The structure and sequence of the hairpin test molecule isshown in FIG. 18A (SEQ ID NO: 15). The oligonucleotide (labeled “primer”in FIG. 18A, SEQ ID NO: 22) is shown annealed to its complementarysequence on the 3′ arm of the hairpin test molecule. The hairpin testmolecule was single-end labeled with ³²P using a labeled T7 promoterprimer in a polymerase chain reaction. The label is present on the 5′arm of the hairpin test molecule and is represented by the star in FIG.18A.

[0708] The cleavage reaction was performed by adding 10 fmoles ofheat-denatured, end-labeled hairpin test molecule, 0.2 μM of the primeroligonucleotide (complementary to the 3′ arm of the hairpin), 50 μM ofeach dNTP and 0.5 units of DNAPTaq (Perkin Elmer) or 0.5 μl of extractcontaining a 5′ nuclease (prepared as described above) in a total volumeof 10 μl in a buffer containing 10 mM Tris-Cl, pH 8.5, 50 mM KCl and 1.5mM MgCl₂. Reactions shown in lanes 3, 5 and 7 were run in the absence ofdNTPs.

[0709] Reactions were incubated at 55° C. for 4 minutes. Reactions werestopped at 55° C. by the addition of 8 μl of 95% formamide with 20 mMEDTA and 0.05% marker dyes per 10 μl reaction volume. Samples were notheated before loading onto denaturing polyacrylamide gels (10%polyacrylamide, 19:1 crosslinking, 7 M urea, 89 mM Tris-borate, pH 8.3,2.8 mM EDTA). The samples were not heated to allow for the resolution ofsingle-stranded and re-duplexed uncleaved hairpin molecules.

[0710]FIG. 18B shows that altered polymerases lacking any detectablesynthetic activity cleave a hairpin structure when an oligonucleotide isannealed to the single-stranded 3′ arm of the hairpin to yield a singlespecies of cleaved product (FIG. 18B, lanes 3 and 4). 5′ nucleases, suchas clone 3D, shown in lanes 3 and 4, produce a single cleaved producteven in the presence of dNTPs. 5′ nucleases that retain a residualamount of synthetic activity (less than 1% of wild type activity)produce multiple cleavage products as the polymerase can extend theoligonucleotide annealed to the 3′ arm of the hairpin thereby moving thesite of cleavage (clone 3B, lanes 5 and 6). Native DNATaq produces evenmore species of cleavage products than do mutant polymerases retainingresidual synthetic activity and additionally converts the hairpinstructure to a double-stranded form in the presence of dNTPs due to thehigh level of synthetic activity in the native polymerase (FIG. 18B,lane 8).

Example 4

[0711] Cleavage of Linear Nucleic Acid Substrates

[0712] From the above, it should be clear that native (i.e., “wildtype”) thermostable DNA polymerases are capable of cleaving hairpinstructures in a specific manner and that this discovery can be appliedwith success to a detection assay. In this example, the mutant DNAPs ofthe present invention are tested against three different cleavagestructures shown in FIG. 20A. Structure 1 in FIG. 20A is simply singlestranded 206-mer (the preparation and sequence information for which wasdiscussed in Example 1C). Structures 2 and 3 are duplexes; structure 2is the same hairpin structure as shown in FIG. 11A (bottom), whilestructure 3 has the hairpin portion of structure 2 removed.

[0713] The cleavage reactions comprised 0.01 pmoles of the resultingsubstrate DNA, and 1 pmole of pilot oligonucleotide in a total volume of10 μl of 10 mM Tris-Cl, pH 8.3, 100 mM KCl, 1 mM MgCl₂. Reactions wereincubated for 30 minutes at 55° C., and stopped by the addition of 8 μlof 95% formamide with 20 mM EDTA and 0.05% marker dyes. Samples wereheated to 75° C. for 2 minutes immediately before electrophoresisthrough a 10% polyacrylamide gel (19:1 cross link), with 7M urea, in abuffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

[0714] The results were visualized by autoradiography and are shown inFIG. 20B with the enzymes indicated as follows: I is native Taq DNAP; IIis native Tfl DNAP; III is CLEAVASE BX shown in FIG. 3E; IV is CLEAVASEBB shown in FIG. 3F; V is the mutant shown in FIG. 4B; and VI isCLEAVASE BN shown in FIG. 3G.

[0715] Structure 2 was used to “normalize” the comparison. For example,it was found that it took 50 ng of Taq DNAP and 300 ng of CLEAVASE BN togive similar amounts of cleavage of Structure 2 in thirty (30) minutes.Under these conditions native Taq DNAP is unable to cleave Structure 3to any significant degree. Native Tfl DNAP cleaves Structure 3 in amanner that creates multiple products.

[0716] By contrast, all of the mutants tested cleave the linear duplexof Structure 3. This finding indicates that this characteristic of themutant DNA polymerases is consistent of thermostable polymerases acrossthermophilic species.

Example 5

[0717] 5′ Exonucleolytic Cleavage (“Nibbling”) by Thermostable DNAPs

[0718] It has been found that thermostable DNAPs, including those of thepresent invention, have a true 5′ exonuclease capable of nibbling the 5′end of a linear duplex nucleic acid structures. In this Example, the 206base pair DNA duplex substrate is again employed (See, Example 1C). Inthis case, it was produced by the use of one ³²P-labeled primer and oneunlabeled primer in a polymerase chain reaction. The cleavage reactionscomprised 0.01 pmoles of heat-denatured, end-labeled substrate DNA (withthe unlabeled strand also present), 5 pmoles of pilot oligonucleotide(see pilot oligos in FIG. 11A) and 0.5 units of DNAPTaq or 0.5μ ofCLEAVASE BB in the E. coli extract (see above), in a total volume of 10μl of 10 mM Tris·Cl, pH 8.5, 50 mM KCl, 1.5 mM MgCl₂.

[0719] Reactions were initiated at 65° C. by the addition of pre-warmedenzyme, then shifted to the final incubation temperature for 30 minutes.The results are shown in FIG. 21A. Samples in lanes 1-4 are the resultswith native Taq DNAP, while lanes 5-8 shown the results with CLEAVASEBB. The reactions for lanes 1, 2, 5, and 6 were performed at 65° C. andreactions for lanes 3, 4, 7, and 8 were performed at 50° C. and all werestopped at temperature by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. Samples were heated to 75° C. for 2minutes immediately before electrophoresis through a 10% acrylamide gel(19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris.Borate, pH8.3, 1.4 mM EDTA. The expected product in reactions 1, 2, 5, and 6 is 85nucleotides long; in reactions 3 and 7, the expected product is 27nucleotides long. Reactions 4 and 8 were performed without pilot, andshould remain at 206 nucleotides. The faint band seen at 24 nucleotidesis residual end-labeled primer from the PCR.

[0720] The surprising result is that CLEAVASE BB under these conditionscauses all of the label to appear in a very small species, suggestingthe possibility that the enzyme completely hydrolyzed the substrate. Todetermine the composition of the fastest-migrating band seen in lanes5-8 (reactions performed with the deletion mutant), samples of the 206base pair duplex were treated with either T7 gene 6 exonuclease (USB) orwith calf intestine alkaline phosphatase (Promega), according tomanufacturers' instructions, to produce either labeled mononucleotide(lane a of FIG. 21B) or free ³²P-labeled inorganic phosphate (lane b ofFIG. 21B), respectively. These products, along with the products seen inlane 7 of panel A were resolved by brief electrophoresis through a 20%acrylamide gel (19:1 cross-link), with 7 M urea, in a buffer of 45 mMTris.Borate, pH 8.3, 1.4 mM EDTA. CLEAVASE BB is thus capable ofconverting the substrate to mononucleotides.

Example 6

[0721] Nibbling is Duplex Dependent

[0722] The nibbling by CLEAVASE BB is duplex dependent. In this Example,internally labeled, single strands of the 206-mer were produced by 15cycles of primer extension incorporating α-³²P labeled dCTP combinedwith all four unlabeled dNTPs, using an unlabeled 206-bp fragment as atemplate. Single and double stranded products were resolved byelectrophoresis through a non-denaturing 6% polyacrylamide gel (29:1cross-link) in a buffer of 45 mM Tris.Borate, pH 8.3, 1.4 mM EDTA,visualized by autoradiography, excised from the gel, eluted by passivediffusion, and concentrated by ethanol precipitation.

[0723] The cleavage reactions comprised 0.04 pmoles of substrate DNA,and 2 μl of CLEAVASE BB (in an E. coli extract as described above) in atotal volume of 40 μl of 10 mM Tris.Cl, pH 8.5, 50 mM KCl, 1.5 mM MgCl₂.Reactions were initiated by the addition of pre-warmed enzyme; 10 μlaliquots were removed at 5, 10, 20, and 30 minutes, and transferred toprepared tubes containing 8 μl of 95% formamide with 30 mM EDTA and0.05% marker dyes. Samples were heated to 75° C. for 2 minutesimmediately before electrophoresis through a 10% acrylamide gel (19: 1cross-linked), with 7 M urea, in a buffer of 45 mM Tris.Borate, pH 8.3,1.4 mM EDTA. Results were visualized by autoradiography as shown in FIG.22. Clearly, the cleavage by CLEAVASE BB depends on a duplex structure;no cleavage of the single strand structure is detected whereas cleavageof the 206-mer duplex is complete.

Example 7

[0724] Nibbling can be Target Directed

[0725] The nibbling activity of the DNAPs of the present invention canbe employed with success in a detection assay. One embodiment of such anassay is shown in FIG. 23. In this assay, a labeled oligo is employedthat is specific for a target sequence. The oligo is in excess of thetarget so that hybridization is rapid. In this embodiment, the oligocontains two fluorescein labels whose proximity on the oligo causestheir emission to be quenched. When the DNAP is permitted to nibble theoligo the labels separate and are detectable. The shortened duplex isdestabilized and disassociates. Importantly, the target is now free toreact with an intact labeled oligo. The reaction can continue until thedesired level of detection is achieved. An analogous, althoughdifferent, type of cycling assay has been described employing lambdaexonuclease. See C. G. Copley and C. Boot, BioTechniques 13:888 (1992).

[0726] The success of such an assay depends on specificity. In otherwords, the oligo must hybridize to the specific target. It is alsopreferred that the assay be sensitive; the oligo ideally should be ableto detect small amounts of target. FIG. 24A shows a 5′-end ³²P-labeledprimer bound to a plasmid target sequence. In this case, the plasmid waspUC 19 (commercially available) which was heat denatured by boiling two(2) minutes and then quick chilling. The primer is a 21-mer (SEQ ID NO:28). The enzyme employed was CLEAVASE BX (a dilution equivalent to5×10⁻³ μl extract) in 100 mM KCl, 10 mM Tris-Cl, pH 8.3, 2 mM MnCl₂. Thereaction was performed at 55° C. for sixteen (16) hours with or withoutgenomic background DNA (from chicken blood). The reaction was stopped bythe addition of 8 μl of 95% formamide with 20 mM EDTA and marker dyes.

[0727] The products of the reaction were resolved by PAGE (10%polyacrylamide, 19:1 cross link, 1×TBE) as seen in FIG. 24B. Lane “M”contains the labeled 21-mer. Lanes 1-3 contain no specific target,although Lanes 2 and 3 contain 100 ng and 200 ng of genomic DNA,respectively. Lanes 4, 5 and 6 all contain specific target with either 0ng, 100 ng, or 200 ng of genomic DNA, respectively. It is clear thatconversion to mononucleotides occurs in Lanes 4, 5 and 6 regardless ofthe presence or amount of background DNA. Thus, the nibbling can betarget directed and specific.

Example 8

[0728] Cleavase Purification

[0729] As noted above, expressed thermostable proteins (i.e., the 5′nucleases), were isolated by crude bacterial cell extracts. Theprecipitated E. coli proteins were then, along with other cell debris,removed by centrifugation. In this Example, cells expressing the BNclone were cultured and collected (500 grams). For each gram (wetweight) of E. coli, 3 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mMEDTA, 100 μM NaCl) was added. The cells were lysed with 200 μg/mllysozyme at room temperature for 20 minutes. Thereafter deoxycholic acidwas added to make a 0.2% final concentration and the mixture wasincubated 15 minutes at room temperature.

[0730] The lysate was sonicated for approximately 6-8 minutes at 0° C.The precipitate was removed by centrifugation (39,000 g for 20 minutes).Polyethyleneimine was added (0.5%) to the supernatant and the mixturewas incubated on ice for 15 minutes. The mixture was centrifuged (5,000g for 15 minutes) and the supernatant was retained. This was heated for30 minutes at 60° C. and then centrifuged again (5,000 g for 15 minutes)and the supernatant was again retained.

[0731] The supernatant was precipitated with 35% animonium sulfate at 4°C. for 15 minutes. The mixture was then centrifuged (5,000 g for 15minutes) and the supernatant was removed. The precipitate was thendissolved in 0.25M KCl, 20 Tris pH 7.6, 0.2% Tween and 0.1 EDTA) andthen dialyzed against Binding Buffer (8× Binding Buffer comprises: 40 mMimidazole, 4M NaCl, 160 mM Tris-HCl, pH 7.9).

[0732] The solubilized protein is then purified on the Ni⁺⁺ column(Novagen). The Binding Buffer is allows to drain to the top of thecolumn bed and load the column with the prepared extract. A flow rate ofabout 10 column volumes per hour is optimal for efficient purification.If the flow rate is too fast, more impurities will contaminate theeluted fraction.

[0733] The column is washed with 25 ml (10 volumes) of 1× Binding Bufferand then washed with 15 ml (6 volumes) of 1× Wash Buffer (8× Wash Buffercomprises: 480 mM imidazole, 4 M NaCl, 160 mM Tris-HCl, pH 7.9). Thebound protein was eluted with 15 ml (6 volumes) of 1× Elute Buffer (4×Elute Buffer comprises: 4 mM imidazole, 2 M NaCl, 80 mM Tris-HCl, pH7.9). Protein is then reprecipitated with 35% ammonium sulfate as above.The precipitate was then dissolved and dialyzed against: 20 mM Tris, 100mM KCl, 1 mM EDTA). The solution was brought up to 0.1% each of Tween 20and NP-40 and stored at 4° C.

Example 9

[0734] The Use of Various Divalent Cations in the Cleavage ReactionInfluences the Nature of the Resulting Cleavage Products

[0735] In comparing the 5′ nucleases generated by the modificationand/or deletion of the C-terminal polymerization domain of Thermusaquaticus DNA polymerase (DNAPTaq), as diagrammed in FIG. 3B-G,significant differences in the strength of the interactions of theseproteins with the 3′ end of primers located upstream of the cleavagesite (as depicted in FIG. 5) were noted. In describing the cleavage ofthese structures by Pol I-type DNA polymerases (See, Example 1, andLyamichev et al., Science 260:778 [1993]), it was observed that in theabsence of a primer, the location of the junction between thedouble-stranded region and the single-stranded 5′ and 3′ arms determinedthe site of cleavage, but in the presence of a primer, the location ofthe 3′ end of the primer became the determining factor for the site ofcleavage. It was postulated that this affinity for the 3′ end was inaccord with the synthesizing function of the DNA polymerase.

[0736] Structure 2, shown in FIG. 20A, was used to test the effects of a3′ end proximal to the cleavage site in cleavage reactions comprisingseveral different solutions (e.g., solutions containing different salts[KCl or NaCl], different divalent cations [Mn²⁺ or Mg²⁺], etc.) as wellas the use of different temperatures for the cleavage reaction. When thereaction conditions were such that the binding of the enzyme (e.g., aDNAP comprising a 5′ nuclease, a modified DNAP or a 5′ nuclease) to the3′ end (of the pilot oligonucleotide) near the cleavage site was strong,the structure shown is cleaved at the site indicated in FIG. 20A. Thiscleavage releases the unpaired 5′ arm and leaves a nick between theremaining portion of the target nucleic acid and the folded 3′ end ofthe pilot oligonucleotide. In contrast, when the reaction conditions aresuch that the binding of the DNAP (comprising a 5′ nuclease) to the 3′end was weak, the initial cleavage was as described above, but after therelease of the 5′ arm, the remaining duplex is digested by theexonuclease function of the DNAP.

[0737] One way of weakening the binding of the DNAP to the 3′ end is toremove all or part of the domain to which at least some of this functionhas been attributed. Some of 5′ nucleases created by deletion of thepolymerization domain of DNAPTaq have enhanced true exonucleasefunction, as demonstrated in Example 5.

[0738] The affinity of these types of enzymes (i.e., 5′ nucleasesassociated with or derived from DNAPs) for recessed 3′ ends may also beaffected by the identity of the divalent cation present in the cleavagereaction. It was demonstrated by Longley et al. (Nucl. Acids Res.,18:7317 [1990]) that the use of MnCl₂ in a reaction with DNAPTaq enabledthe polymerase to remove nucleotides from the 5′ end of a primerannealed to a template, albeit inefficiently. Similarly, by examinationof the cleavage products generated using Structure 2 from FIG. 20A, asdescribed above, in a reaction containing either DNAPTaq or the CLEAVASEBB nuclease, it was observed that the substitution of MnCl₂ for MgCl₂ inthe cleavage reaction resulted in the exonucleolytic “nibbling” of theduplex downstream of the initial cleavage site. While not limiting theinvention to any particular mechanism, it is thought that thesubstitution of MnCl₂ for MgCl₂ in the cleavage reaction lessens theaffinity of these enzymes for recessed 3′ ends.

[0739] In all cases, the use of MnCl₂ enhances the 5′ nuclease function,and in the case of the CLEAVASE BB nuclease, a 50- to 100-foldstimulation of the 5′ nuclease function is seen. Thus, while theexonuclease activity of these enzymes was demonstrated above in thepresence of MgCl₂, the assays described below show a comparable amountof exonuclease activity using 50 to 100-fold less enzyme when MnCl₂ isused in place of MgCl₂. When these reduced amounts of enzyme are used ina reaction mixture containing MgCl₂, the nibbling or exonucleaseactivity is much less apparent than that seen in Examples 5-7.

[0740] Similar effects are observed in the performance of the nucleicacid detection assay described in Examples 10-39 below when reactionsperformed in the presence of either MgCl₂ or MnCl₂ are compared. In thepresence of either divalent cation, the presence of the INVADERoligonucleotide (described below) forces the site of cleavage into theprobe duplex, but in the presence of MnCl₂ the probe duplex can befurther nibbled producing a ladder of products that are visible when a3′ end label is present on the probe oligonucleotide. When the INVADERoligonucleotide is omitted from a reaction containing Mn²⁺, the probe isnibbled from the 5′ end. Mg²⁺-based reactions display minimal nibblingof the probe oligonucleotide. In any of these cases, the digestion ofthe probe is dependent upon the presence of the target nucleic acid. Inthe examples below, the ladder produced by the enhanced nibblingactivity observed in the presence of Mn²⁺ is used as a positiveindicator that the probe oligonucleotide has hybridized to the targetsequence.

Example 10

[0741] Invasive 5′ Endonucleolytic Cleavage by Thermostable 5′ Nucleasesin the Absence of Polymerization

[0742] As described in the Examples above, 5′ nucleases cleave near thejunction between single-stranded and base-paired regions in a bifurcatedduplex, usually about one base pair into the base-paired region. In thisExample, it is shown that thermostable 5′ nucleases, including those ofthe present invention (e.g., CLEAVASE BN nuclease, CLEAVASE A/Gnuclease), have the ability to cleave a greater distance into the basepaired region when provided with an upstream oligonucleotide bearing a3′ region that is homologous to a 5′ region of the subject duplex, asshown in FIG. 26.

[0743]FIG. 26 shows a synthetic oligonucleotide that was designed tofold upon itself and that consists of the following sequence:5′-GTTCTCTGCTCTCTGGTCGCTG TCTCGCTTGTGAAACAAGCGAGACAGCGTGGTCTCTCG-3′ (SEQID NO: 29). This oligonucleotide is referred to as the “S-60 Hairpin.”The 15 basepair hairpin formed by this oligonucleotide is furtherstabilized by a “tri-loop” sequence in the loop end (i.e., threenucleotides form the loop portion of the hairpin) (Hiraro et al.,Nucleic Acids Res., 22(4):576 [1994]). FIG. 26 also show the sequence ofthe P-15 oligonucleotide and the location of the region ofcomplementarity shared by the P-15 and S-60 hairpin oligonucleotides.The sequence of the P-15 oligonucleotide is 5′-CGAGAGACCACGCTG-3′ (SEQID NO: 30). As discussed in detail below, the solid black arrowheadsshown in FIG. 26 indicate the sites of cleavage of the S-60 hairpin inthe absence of the P-15 oligonucleotide and the hollow arrow headsindicate the sites of cleavage in the presence of the P-15oligonucleotide. The size of the arrow head indicates the relativeutilization of a particular site.

[0744] The S-60 hairpin molecule was labeled on its 5′ end with biotinfor subsequent detection. The S-60 hairpin was incubated in the presenceof a thermostable 5′ nuclease in the presence or the absence of the P-15oligonucleotide. The presence of the full duplex that can be formed bythe S-60 hairpin is demonstrated by cleavage with the CLEAVASE BN 5′nuclease, in a primer-independent fashion (i.e., in the absence of theP-15 oligonucleotide). The release of 18 and 19-nucleotide fragmentsfrom the 5′ end of the S-60 hairpin molecule showed that the cleavageoccurred near the junction between the single and double strandedregions when nothing is hybridized to the 3′ arm of the S-60 hairpin(FIG. 27, lane 2).

[0745] The reactions shown in FIG. 27 were conducted as follows. Twentyfmole of the 5′ biotin-labeled hairpin DNA (SEQ ID NO: 29) was combinedwith 0.1 ng of CLEAVASE BN enzyme and 1 μl of 100 mM MOPS (pH 7.5)containing 0.5% each of Tween-20 and NP-40 in a total volume of 9 μl. Inthe reaction shown in lane 1, the enzyme was omitted and the volume wasmade up by addition of distilled water (this served as the uncut or noenzyme control). The reaction shown in lane 3 of FIG. 27 also included0.5 pmole of the P15 oligonucleotide (SEQ ID NO: 30), which canhybridize to the unpaired 3′ arm of the S-60 hairpin (SEQ ID NO: 29), asdiagrammed in FIG. 26.

[0746] The reactions were overlaid with a drop of mineral oil, heated to95° C. for 15 seconds, then cooled to 37° C., and the reaction wasstarted by the addition of 1 μl of 10 mM MnCl₂ to each tube. After 5minutes, the reactions were stopped by the addition of 6 μl of 95%formamide containing 20 mM EDTA and 0.05% marker dyes. Samples wereheated to 75° C. for 2 minutes immediately before electrophoresisthrough a 15% acrylamide gel (19:1 cross-linked), with 7 M urea, in abuffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

[0747] After electrophoresis, the gel plates were separated allowing thegel to remain flat on one plate. A 0.2 mm-pore positively-charged nylonmembrane (NYTRAN, Schleicher and Schuell, Keene, N.H.), pre-wetted inH₂O, was laid on top of the exposed gel. All air bubbles were removed.Two pieces of 3MM filter paper (Whatman) were then placed on top of themembrane, the other glass plate was replaced, and the sandwich wasclamped with binder clips. Transfer was allowed to proceed overnight.After transfer, the membrane was carefully peeled from the gel andallowed to air dry. After complete drying, the membrane was washed in1.2× Sequenase Images Blocking Buffer (United States Biochemical) using0.3 ml of buffer/cm² of membrane. The wash was performed for 30 minutesat room temperature. A streptavidin-alkaline phosphatase conjugate(SAAP, United States Biochemical) was added to a 1:4000 dilutiondirectly to the blocking solution, and agitated for 15 minutes. Themembrane was rinsed briefly with H₂O and then washed three times for 5minutes per wash using 0.5 ml/cm² of 1× SAAP buffer (100 mM Tris-HCl, pH10, 50 mM NaCl) with 0.1% sodium dodecyl sulfate (SDS). The membrane wasrinsed briefly with H₂O between each wash. The membrane was then washedonce in 1× SAAP buffer containing 1 mM MgCl₂ without SDS, drainedthoroughly and placed in a plastic heat-sealable bag. Using a sterilepipet, 5 mls of CDP-Star™ (Tropix, Bedford, Mass.) chemiluminescentsubstrate for alkaline phosphatase were added to the bag and distributedover the entire membrane for 2-3 minutes. The CDP-Star™-treated membranewas exposed to XRP X-ray film (Kodak) for an initial exposure of 10minutes.

[0748] The resulting autoradiograph is shown in FIG. 27. In FIG. 27, thelane labeled “M” contains the biotinylated P-15 oligonucleotide, whichserved as a marker. The sizes (in nucleotides) of the uncleaved S-60hairpin (60 nuc; lane 1), the marker (15 nuc; lane “M”) and the cleavageproducts generated by cleavage of the S-60 hairpin in the presence (lane3) or absence (lane 2) of the P-15 oligonucleotide are indicated.

[0749] Because the complementary regions of the S-60 hairpin are locatedon the same molecule, essentially no lag time should be needed to allowhybridization (i.e., to form the duplex region of the hairpin). Thishairpin structure would be expected to form long before the enzyme couldlocate and cleave the molecule. As expected, cleavage in the absence ofthe primer oligonucleotide was at or near the junction between theduplex and single-stranded regions, releasing the unpaired 5′ arm (FIG.27, lane 2). The resulting cleavage products were 18 and 19 nucleotidesin length.

[0750] It was expected that stability of the S-60 hairpin with thetri-loop would prevent the P-15 oligonucleotide from promoting cleavagein the “primer-directed” manner described in Example 1 above, becausethe 3′ end of the “primer” would remain unpaired. Surprisingly, it wasfound that the enzyme seemed to mediate an “invasion” by the P-15 primerinto the duplex region of the S-60 hairpin, as evidenced by the shiftingof the cleavage site 3 to 4 basepairs further into the duplex region,releasing the larger products (22 and 21 nuc.) observed in lane 3 ofFIG. 27.

[0751] The precise sites of cleavage of the S-60 hairpin are diagrammedon the structure in FIG. 26, with the solid black arrowheads indicatingthe sites of cleavage in the absence of the P-15 oligonucleotide and thehollow arrow heads indicating the sites of cleavage in the presence ofP-15.

[0752] These data show that the presence on the 3′ arm of anoligonucleotide having some sequence homology with the first severalbases of the similarly oriented strand of the downstream duplex can be adominant factor in determining the site of cleavage by 5′ nucleases.Because the oligonucleotide that shares some sequence homology with thefirst several bases of the similarly oriented strand of the downstreamduplex appears to invade the duplex region of the hairpin, it isreferred to as an “INVADER” oligonucleotide. As shown in the Examplesbelow, an INVADER oligonucleotide appears to invade (or displace) aregion of duplexed nucleic acid regardless of whether the duplex regionis present on the same molecule (i.e., a hairpin) or whether the duplexis formed between two separate nucleic acid strands.

Example 11

[0753] The INVADER Oligonucleotide Shifts the Site of Cleavage in aPre-Formed Probe/Target Duplex

[0754] In Example 10, it was demonstrated that an INVADERoligonucleotide could shift the site at which a 5′ nuclease cleaves aduplex region present on a hairpin molecule. In this Example, theability of an INVADER oligonucleotide to shift the site of cleavagewithin a duplex region formed between two separate strands of nucleicacid molecules was examined.

[0755] A single-stranded target DNA comprising the single-strandedcircular M13mp 19 molecule and a labeled (fluorescein) probeoligonucleotide were mixed in the presence of the reaction buffercontaining salt (KCl) and divalent cations (Mg²⁺ or Mn²⁺) to promoteduplex formation. The probe oligonucleotide refers to a labeledoligonucleotide that is complementary to a region along the targetmolecule (e.g., M13mp19). A second oligonucleotide (unlabeled) was addedto the reaction after the probe and target had been allowed to anneal.The second oligonucleotide binds to a region of the target that islocated downstream of the region to which the probe oligonucleotidebinds. This second oligonucleotide contains sequences that arecomplementary to a second region of the target molecule. If the secondoligonucleotide contains a region that is complementary to a portion ofthe sequences along the target to which the probe oligonucleotide alsobinds, this second oligonucleotide is referred to as an INVADERoligonucleotide (see FIG. 28c).

[0756]FIG. 32 depicts the annealing of two oligonucleotides to regionsalong the M13mp19 target molecule (bottom strand in all three structuresshown). In FIG. 28 only a 52 nucleotide portion of the M13mp19 moleculeis shown; this 52 nucleotide sequence is listed in SEQ ID NO: 31. Theprobe oligonucleotide contains a fluorescein label at the 3′ end; thesequence of the probe is 5′-AGAAAGGAAGGGAAGAAAGCGAAAGG-3′ (SEQ ID NO:32). In FIG. 28, sequences comprising the second oligonucleotide,including the INVADER oligonucleotide are underlined. In FIG. 28a, thesecond oligonucleotide, which has the sequence5′-GACGGGGAAAGCCGGCGAACG-3′ (SEQ ID NO: 33), is complementary to adifferent and downstream region of the target molecule than is the probeoligonucleotide (labeled with fluorescein or “Fluor”); there is a gapbetween the second, upstream oligonucleotide and the probe for thestructure shown in FIG. 28a. In FIG. 28b, the second, upstreamoligonucleotide, which has the sequence 5′-GAAAGCCGGCGAACGTGGCG-3′ (SEQID NO: 34), is complementary to a different region of the targetmolecule than is the probe oligonucleotide, but in this case, the secondoligonucleotide and the probe oligonucleotide abut one another (that isthe 3′ end of the second, upstream oligonucleotide is immediatelyadjacent to the 5′ end of the probe such that no gap exists betweenthese two oligonucleotides). In FIG. 28c, the second, upstreamoligonucleotide (5′-GGCGAACGTGGCGAGAAAGGA-3′ [SEQ ID NO: 35]) and theprobe oligonucleotide share a region of complementarity with the targetmolecule. Thus, the upstream oligonucleotide has a 3′ arm that has asequence identical to the first several bases of the downstream probe.In this situation, the upstream oligonucleotide is referred to as an“INVADER” oligonucleotide.

[0757] The effect of the presence of an INVADER oligonucleotide upon thepattern of cleavage in a probe/target duplex formed prior to theaddition of the INVADER was examined. The INVADER oligonucleotide andthe enzyme were added after the probe was allowed to anneal to thetarget and the position and extent of cleavage of the probe wereexamined to determine a) if the INVADER was able to shift the cleavagesite to a specific internal region of the probe, and b), if the reactioncould accumulate specific cleavage products over time, even in theabsence of thermal cycling, polymerization, or exonuclease removal ofthe probe sequence.

[0758] The reactions were carried out as follows. Twenty μl each of twoenzyme mixtures were prepared, containing 2 μl of CLEAVASE A/G nucleaseextract (prepared as described in Example 2), with or without 50 pmoleof the INVADER oligonucleotide (SEQ ID NO: 35), as indicated, per 4 μlof the mixture. For each of the eight reactions shown in FIG. 29, 150fmole of M13mp19 single-stranded DNA (available from Life Technologies,Inc.) was combined with 5 pmoles of fluorescein labeled probe (SEQ IDNO: 32), to create the structure shown in FIG. 28c, but without theINVADER oligonucleotide present (the probe/target mixture). One half (4tubes) of the probe/target mixtures were combined with 1 μl of 100 mMMOPS, pH 7.5 with 0.5% each of Tween-20 and NP-40, 0.5 μl of 1 M KCl and0.25 μl of 80 mM MnCl₂, and distilled water to a volume of 6 μl. Thesecond set of probe/target mixtures were combined with 1 μl of 100 mMMOPS, pH 7.5 with 0.5% each of Tween-20 and NP-40, 0.5 μl of 1 M KCl and0.25 μl of 80 mM MgCl₂. The second set of mixtures therefore containedMgCl₂ in place of the MnCl₂ present in the first set of mixtures.

[0759] The mixtures (containing the probe/target with buffer, KCl anddivalent cation) were covered with a drop of CHILLOUT evaporationbarrier and were brought to 60° C. for 5 minutes to allow annealing.Four μl of the above enzyme mixtures without the INVADER oligonucleotidewas added to reactions whose products are shown in lanes 1, 3, 5 and 7of FIG. 29. Reactions whose products are shown lanes 2, 4, 6, and 8 ofFIG. 29 received the same amount of enzyme mixed with the INVADERoligonucleotide (SEQ ID NO: 35). Reactions 1, 2, 5 and 6 were incubatedfor 5 minutes at 60° C. and reactions 3, 4, 7 and 8 were incubated for15 minutes at 60° C.

[0760] All reactions were stopped by the addition of 8 μl of 95%formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to90° C. for 1 minute immediately before electrophoresis through a 20%acrylamide gel (19:1 cross-linked), containing 7 M urea, in a buffer of45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Following electrophoresis, thereaction products and were visualized by the use of an Hitachi FMBIOfluorescence imager, the output of which is seen in FIG. 29. The verylow molecular weight fluorescent material seen in all lanes at or nearthe salt front in FIG. 29 and other fluoro-imager Figures is observedwhen fluorescently-labeled oligonucleotides are electrophoresed andimaged on a fluoro-imager. This material is not a product of thecleavage reaction.

[0761] The use of MnCl₂ in these reactions (lanes 1-4) stimulates thetrue exonuclease or “nibbling” activity of the CLEAVASE enzyme, asdescribed in Example 6, as is clearly seen in lanes 1 and 3 of FIG. 29.This nibbling of the probe oligonucleotide (SEQ ID NO: 32) in theabsence of INVADER oligonucleotide (SEQ ID NO: 35) confirms that theprobe oligonucleotide is forming a duplex with the target sequence. Theladder-like products produced by this nibbling reaction may be difficultto differentiate from degradation of the probe by nucleases that mightbe present in a clinical specimen. In contrast, introduction of theINVADER oligonucleotide (SEQ ID NO: 35) caused a distinctive shift inthe cleavage of the probe, pushing the site of cleavage 6 to 7 basesinto the probe, confirming the annealing of both oligonucleotides. Inpresence of MnCl₂, the exonuclease “nibbling” may occur after theINVADER-directed cleavage event, until the residual duplex isdestabilized and falls apart.

[0762] In a magnesium based cleavage reaction (lanes 5-8), the nibblingor true exonuclease function of the CLEAVASE A/G is enzyme suppressed(but the endonucleolytic function of the enzyme is essentiallyunaltered), so the probe oligonucleotide is not degraded in the absenceof the INVADER (FIG. 29, lanes 5 and 7). When the INVADER is added, itis clear that the INVADER oligonucleotide can promote a shift in thesite of the endonucleolytic cleavage of the annealed probe. Comparisonof the products of the 5 and 15 minute reactions with INVADER (lanes 6and 8 in FIG. 29) shows that additional probe hybridizes to the targetand is cleaved. The calculated melting temperature (T_(m)) of theportion of probe that is not invaded (i.e., nucleotides 9-26 of SEQ IDNO: 32) is 56° C., so the observed turnover (as evidenced by theaccumulation of cleavage products with increasing reaction time)suggests that the full length of the probe molecule, with a calculatedT_(m) of 76° C., is must be involved in the subsequent probe annealingevents in this 60° C. reaction.

Example 12

[0763] The Overlap of the 3′ INVADER Oligonucleotide Sequence with the5′ Region of the Probe Causes a Shift in the Site of Cleavage

[0764] In Example 11, the ability of an INVADER oligonucleotide to causea shift in the site of cleavage of a probe annealed to a target moleculewas demonstrated. In this Example, experiments were conducted to examinewhether the presence of an oligonucleotide upstream from the probe wassufficient to cause a shift in the cleavage site(s) along the probe orwhether the presence of nucleotides on the 3′ end of the INVADERoligonucleotide that have the same sequence as the first severalnucleotides at the 5′ end of the probe oligonucleotide were required topromote the shift in cleavage.

[0765] To examine this point, the products of cleavage obtained fromthree different arrangements of target-specific oligonucleotides arecompared. A diagram of these oligonucleotides and the way in which theyhybridize to a test nucleic acid, M13mp19, is shown in FIG. 28. In FIG.28a, the 3′ end of the upstream oligonucleotide (SEQ ID NO: 33) islocated upstream of the 5′ end of the downstream “probe” oligonucleotide(SEQ ID NO: 32) such that a region of the M13 target that is not pairedto either oligonucleotide is present. In FIG. 28b, the sequence of theupstream oligonucleotide (SEQ ID NO: 34) is immediately upstream of theprobe (SEQ ID NO: 32), having neither a gap nor an overlap between thesequences. FIG. 28c diagrams the arrangement of the substrates used inthe assay of the present invention, showing that the upstream “INVADER”oligonucleotide (SEQ ID NO: 35) has the same sequence on a portion ofits 3′ region as that present in the 5′ region of the downstream probe(SEQ ID NO: 32). That is to say, these regions will compete to hybridizeto the same segment of the M13 target nucleic acid.

[0766] In these experiments, four enzyme mixtures were prepared asfollows (planning 5 μl per digest): Mixture 1 contained 2.25 μl ofCLEAVASE A/G nuclease extract (prepared as described in Example 2) per 5μl of mixture, in 20 mM MOPS, pH 7.5 with 0.1 % each of Tween 20 andNP-40, 4 mM MnCl₂ and 100 mM KCl. Mixture 2 contained 11.25 units of TaqDNA polymerase (Promega) per 5 μl of mixture in 20 mM MOPS, pH 7.5 with0.1 % each of Tween 20 and NP-40, 4 mM MnCl₂ and 100 mM KCl. Mixture 3contained 2.25 μl of CLEAVASE A/G nuclease extract per 5 μl of mixturein 20 mM Tris-HCl, pH 8.5, 4 mM MgCl₂ and 100 mM KCl. Mixture 4contained 11.25 units of Taq DNA polymerase per 5 μl of mixture in 20 mMTris-HCl, pH 8.5, 4 mM MgCl₂ and 100 mM KCl.

[0767] For each reaction, 50 fmole of M13mp19 single-stranded DNA (thetarget nucleic acid) was combined with 5 pmole of the probeoligonucleotide (SEQ ID NO: 32 which contained a fluorescein label atthe 3′ end) and 50 pmole of one of the three upstream oligonucleotidesdiagrammed in FIG. 28 (i.e., one of SEQ ID NOS: 33-35), in a totalvolume of 5 μl of distilled water. The reactions were overlaid with adrop of ChillOut™ evaporation barrier and warmed to 62° C. The cleavagereactions were started by the addition of 5 μl of an enzyme mixture toeach tube, and the reactions were incubated at 62° C. for 30 min. Thereactions shown in lanes 1-3 of FIG. 30 received Mixture 1; reactions4-6 received Mixture 2; reactions 7-9 received Mixture 3 and reactions10-12 received Mixture 4.

[0768] After 30 minutes at 62° C., the reactions were stopped by theaddition of 8 μl of 95% formamide with 20 mM EDTA and 0.05% marker dyes.Samples were heated to 75° C. for 2 minutes immediately beforeelectrophoresis through a 20% acrylamide gel (19:1 cross-linked), with 7M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

[0769] Following electrophoresis, the products of the reactions werevisualized by the use of an Hitachi FMBIO fluorescence imager, theoutput of which is seen in FIG. 30. The reaction products shown in lanes1, 4, 7 and 10 of FIG. 30 were from reactions that contained SEQ ID NO:33 as the upstream oligonucleotide (see FIG. 28a). The reaction productsshown in lanes 2, 5, 8 and 11 of FIG. 30 were from reactions thatcontained SEQ ID NO: 34 as the upstream oligonucleotide (see FIG. 28b).The reaction products shown in lanes 3, 6, 9 and 12 of FIG. 30 were fromreactions that contained SEQ ID NO: 35, the INVADER oligonucleotide, asthe upstream oligonucleotide (see FIG. 28c).

[0770] Examination of the Mn²⁺ based reactions using either CLEAVASE A/Gnuclease or DNAPTaq as the cleavage agent (lanes 1 through 3 and 4through 6, respectively) shows that both enzymes have active exonucleasefunction in these buffer conditions. The use of a 3′ label on the probeoligonucleotide allows the products of the nibbling activity to remainlabeled, and therefore visible in this assay. The ladders seen in lanes1, 2, 4 and 5 confirm that the probe hybridize to the target DNA asintended. These lanes also show that the location of the non-invasiveoligonucleotides have little effect on the products generated. Theuniform ladder created by these digests would be difficult todistinguish from a ladder causes by a contaminating nuclease, as onemight find in a clinical specimen. In contrast, the products displayedin lanes 3 and 6, where an INVADER oligonucleotide was provided todirect the cleavage, show a very distinctive shift, so that the primarycleavage product is smaller than those seen in the non-invasivecleavage. This product is then subject to further nibbling in theseconditions, as indicated by the shorter products in these lanes. TheseINVADER-directed cleavage products would be easily distinguished from abackground of non-specific degradation of the probe oligonucleotide.

[0771] When Mg²⁺ is used as the divalent cation the results are evenmore distinctive. In lanes 7, 8, 10 and 11 of FIG. 30, where theupstream oligonucleotides were not invasive, minimal nibbling isobserved. The products in the DNAPTaq reactions show some accumulationof probe that has been shortened on the 5′ end by one or two nucleotidesconsistent with previous examination of the action of this enzyme onnicked substrates (Longley et al., supra). When the upstreamoligonucleotide is invasive, however, the appearance of thedistinctively shifted probe band is seen. These data clearly indicatedthat it is the invasive 3′ portion of the upstream oligonucleotide thatis responsible for fixing the site of cleavage of the downstream probe.

[0772] Thus, the above results demonstrate that it is the presence ofthe free or initially non-annealed nucleotides at the 3′ end of theINVADER oligonucleotide that mediate the shift in the cleavage site, notjust the presence of an oligonucleotide annealed upstream of the probe.Nucleic acid detection assays that employ the use of an INVADERoligonucleotide are termed “INVADER-directed cleavage” assays.

Example 13

[0773] INVADER-Directed Cleavage Recognizes Single and Double StrandedTarget Molecules in a Background of Non-Target DNA Molecules

[0774] For a nucleic acid detection method to be broadly useful, it mustbe able to detect a specific target in a sample that may contain largeamounts of other DNA, (e.g., bacterial or human chromosomal DNA). Theability of the INVADER directed cleavage assay to recognize and cleaveeither single- or double-stranded target molecules in the presence oflarge amounts of non-target DNA was examined. In these experiments amodel target nucleic acid, M13, in either single or double stranded form(single-stranded M13mp18 is available from Life Technologies, Inc anddouble-stranded M13mp19 is available from NEB), was combined with humangenomic DNA (Novagen) and then utilized in INVADER-directed cleavagereactions. Before the start of the cleavage reaction, the DNAs wereheated to 95° C. for 15 minutes to completely denature the samples, asis standard practice in assays, such as polymerase chain reaction orenzymatic DNA sequencing, which involve solution hybridization ofoligonucleotides to double-stranded target molecules.

[0775] For each of the reactions shown in lanes 2-5 of FIG. 31, thetarget DNA (25 fmole of the ss DNA or 1 pmole of the ds DNA) wascombined with 50 pmole of the INVADER oligonucleotide (SEQ ID NO: 35);for the reaction shown in lane 1 the target DNA was omitted. Reactions1, 3 and 5 also contained 470 ng of human genomic DNA. These mixtureswere brought to a volume of 10 μl with distilled water, overlaid with adrop of ChillOut™ evaporation barrier, and brought to 95° C. for 15minutes. After this incubation period, and still at 95° C., each tubereceived 10 μl of a mixture comprising 2.25 μl of CLEAVASE A/G nucleaseextract (prepared as described in Example 2) and 5 pmole of the probeoligonucleotide (SEQ ID NO: 32), in 20 mM MOPS, pH 7.5 with 0.1 % eachof Tween 20 and NP-40, 4 mM MnCl₂ and 100 mM KCl. The reactions werebrought to 62° C. for 15 minutes and stopped by the addition of 12 μl of95% formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heatedto 75° C. for 2 minutes immediately before electrophoresis through a 20%acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. The products of the reactions werevisualized by the use of an Hitachi FMBIO fluorescence imager. Theresults are displayed in FIG. 31.

[0776] In FIG. 31, lane 1 contains the products of the reactioncontaining the probe (SEQ ID NO: 32), the INVADER oligonucleotide (SEQID NO: 35) and human genomic DNA. Examination of lane 1 shows that theprobe and INVADER oligonucleotides are specific for the target sequence,and that the presence of genomic DNA does not cause any significantbackground cleavage.

[0777] In FIG. 31, lanes 2 and 3 contain reaction products fromreactions containing the single-stranded target DNA (M13mp18), the probe(SEQ ID NO: 32) and the INVADER oligonucleotide (SEQ ID NO: 35) in theabsence or presence of human genomic DNA, respectively. Examination oflanes 2 and 3 demonstrate that the INVADER detection assay may be usedto detect the presence of a specific sequence on a single-strandedtarget molecule in the presence or absence of a large excess ofcompetitor DNA (human genomic DNA).

[0778] In FIG. 31, lanes 4 and 5 contain reaction products fromreactions containing the double-stranded target DNA (M13mp19), the probe(SEQ ID NO: 32) and the INVADER oligonucleotide (SEQ ID NO: 35) in theabsence or presence of human genomic DNA, respectively. Examination oflanes 4 and 5 show that double stranded target molecules are eminentlysuitable for INVADER-directed detection reactions. The success of thisreaction using a short duplexed molecule, M13mp19, as the target in abackground of a large excess of genomic DNA is especially noteworthy asit would be anticipated that the shorter and less complex M13 DNAstrands would be expected to find their complementary strand more easilythan would the strands of the more complex human genomic DNA. If the M13DNA reannealed before the probe and/or INVADER oligonucleotides couldbind to the target sequences along the M13 DNA, the cleavage reactionwould be prevented. In addition, because the denatured genomic DNA wouldpotentially contain regions complementary to the probe and/or INVADERoligonucleotides it was possible that the presence of the genomic DNAwould inhibit the reaction by binding these oligonucleotides therebypreventing their hybridization to the M13 target. The above resultsdemonstrate that these theoretical concerns are not a problem under thereaction conditions employed above.

[0779] In addition to demonstrating that the INVADER detection assay maybe used to detect sequences present in a double-stranded target, thesedata also show that the presence of a large amount of non-target DNA(470 ng/20 μl reaction) does not lessen the specificity of the cleavage.While this amount of DNA does show some impact on the rate of productaccumulation, probably by binding a portion of the enzyme, the nature ofthe target sequence, whether single- or double-stranded nucleic acid,does not limit the application of this assay.

Example 14

[0780] Signal Accumulation in the INVADER-Directed Cleavage Assay as aFunction of Target Concentration

[0781] To investigate whether the INVADER-directed cleavage assay couldbe used to indicate the amount of target nucleic acid in a sample, thefollowing experiment was performed. Cleavage reactions were assembledthat contained an INVADER oligonucleotide (SEQ ID NO: 35), a labeledprobe (SEQ ID NO: 32) and a target nucleic acid, M13mp19. A series ofreactions, which contained smaller and smaller amounts of the M13 targetDNA, was employed in order to examine whether the cleavage productswould accumulate in a manner that reflected the amount of target DNApresent in the reaction.

[0782] The reactions were conducted as follows. A master mix containingenzyme and buffer was assembled. Each 5 μl of the master mixturecontained 25 ng of CLEAVASE BN nuclease in 20 mM MOPS (pH 7.5) with 0.1% each of Tween 20 and NP-40, 4 mM MnCl₂ and 100 mM KCl. For each of thecleavage reactions shown in lanes 4-13 of FIG. 32, a DNA mixture wasgenerated that contained 5 pmoles of the fluorescein-labeled probeoligonucleotide (SEQ ID NO: 32), 50 pmoles of the INVADERoligonucleotide (SEQ ID NO: 35) and 100, 50, 10, 5, 1, 0.5, 0.1, 0.05,0.01 or 0.005 fmoles of single-stranded M13mp19, respectively, for every5 μl of the DNA mixture. The DNA solutions were covered with a drop ofCHILLOUT evaporation barrier and brought to 61° C. The cleavagereactions were started by the addition of 5 μl of the enzyme mixture toeach of tubes (final reaction volume was 10 μl). After 30 minutes at 61°C., the reactions were terminated by the addition of 8 μl of 95%formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to90° C. for 1 minute immediately before electrophoresis through a 20%denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffercontaining 45 mM Tris-Borate (pH 8.3), 1.4 mM EDTA. To provide reference(i.e., standards), 1.0, 0.1 and 0.01 pmole aliquots offluorescein-labeled probe oligonucleotide (SEQ ID NO: 32) were dilutedwith the above formamide solution to a final volume of 18 μl. Thesereference markers were loaded into lanes 1-3, respectively of the gel.The products of the cleavage reactions (as well as the referencestandards) were visualized following electrophoresis by the use of aHitachi FMBIO fluorescence imager. The results are displayed in FIG. 32.

[0783] In FIG. 32, boxes appear around fluorescein-containing nucleicacid (i.e., the cleaved and uncleaved probe molecules) and the amount offluorescein contained within each box is indicated under the box. Thebackground fluorescence of the gel (see box labeled “background”) wassubtracted by the fluoro-imager to generate each value displayed under abox containing cleaved or uncleaved probe products (the boxes arenumbered 1-14 at top left with a V followed by a number below the box).The lane marked “M” contains fluoresceinated oligonucleotides, whichserved as markers.

[0784] The results shown in FIG. 32, demonstrate that the accumulationof cleaved probe molecules in a fixed-length incubation period reflectsthe amount of target DNA present in the reaction. The results alsodemonstrate that the cleaved probe products accumulate in excess of thecopy number of the target. This is clearly demonstrated by comparing theresults shown in lane 3, in which 10 fmole (0.01 pmole) of uncut probeare displayed with the results shown in 5, where the products thataccumulated in response to the presence of 10 fmole of target DNA aredisplayed. These results show that the reaction can cleave hundreds ofprobe oligonucleotide molecules for each target molecule present,dramatically amplifying the target-specific signal generated in theINVADER-directed cleavage reaction.

Example 15

[0785] Effect of Saliva Extract on the INVADER-Directed Cleavage Assay

[0786] For a nucleic acid detection method to be useful in a medical(i.e., a diagnostic) setting, it must not be inhibited by materials andcontaminants likely to be found in a typical clinical specimen. To testthe susceptibility of the INVADER-directed cleavage assay to variousmaterials, including but not limited to nucleic acids, glycoproteins andcarbohydrates, likely to be found in a clinical sample, a sample ofhuman saliva was prepared in a manner consistent with practices in theclinical laboratory and the resulting saliva extract was added to theINVADER-directed cleavage assay. The effect of the saliva extract uponthe inhibition of cleavage and upon the specificity of the cleavagereaction was examined.

[0787] One and one-half milliliters of human saliva were collected andextracted once with an equal volume of a mixture containingphenol:chloroform:isoamyl alcohol (25:24:1). The resulting mixture wascentrifuged in a microcentrifuge to separate the aqueous and organicphases. The upper, aqueous phase was transferred to a fresh tube.One-tenth volumes of 3 M NaOAc were added and the contents of the tubewere mixed. Two volumes of 100% ethyl alcohol were added to the mixtureand the sample was mixed and incubated at room temperature for 15minutes to allow a precipitate to form. The sample was centrifuged in amicrocentrifuge at 13,000 rpm for 5 minutes and the supernatant wasremoved and discarded. A milky pellet was easily visible. The pellet wasrinsed once with 70% ethanol, dried under vacuum and dissolved in 200 μlof 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA (this constitutes the salivaextract). Each μl of the saliva extract was equivalent to 7.5 μl ofsaliva. Analysis of the saliva extract by scanning ultravioletspectrophotometry showed a peak absorbance at about 260 nm and indicatedthe presence of approximately 45 ng of total nucleic acid per μl ofextract.

[0788] The effect of the presence of saliva extract upon the followingenzymes was examined: CLEAVASE BN nuclease, CLEAVASE A/G nuclease andthree different lots of DNAPTaq: AmpliTaq® (Perkin Elmer; a recombinantform of DNAPTaq), AmpliTaq® LD (Perkin-Elmer; a recombinant DNAPTaqpreparation containing very low levels of DNA) and Taq DNA polymerase(Fischer). For each enzyme tested, an enzyme/probe mixture was madecomprising the chosen amount of enzyme with 5 pmole of the probeoligonucleotide (SEQ ID NO: 32) in 10 μl of 20 mM MOPS (pH 7.5)containing 0.1% each of Tween 20 and NP-40, 4 mM MnCl₂, 100 mM KCl and100 μg/ml BSA. The following amounts of enzyme were used: 25 ng ofCLEAVASE BN prepared as described in Example 8; 2 μl of CLEAVASE A/Gnuclease extract prepared as described in Example 2; 2.25 μl (11.25polymerase units) the following DNA polymerases: AmpliTaq® DNApolymerase (Perkin Elmer); AmpliTaq® DNA polymerase LD (low DNA; fromPerkin Elmer); Taq DNA polymerase (Fisher Scientific).

[0789] For each of the reactions shown in FIG. 33, except for that shownin lane 1, the target DNA (50 fmoles of single-stranded M13mp19 DNA) wascombined with 50 pmole of the INVADER oligonucleotide (SEQ ID NO: 35)and 5 pmole of the probe oligonucleotide (SEQ ID NO: 32); target DNA wasomitted in reaction 1 (lane 1). Reactions 1, 3, 5, 7, 9 and 11 included1.5 μl of saliva extract. These mixtures were brought to a volume of 5μl with distilled water, overlaid with a drop of CHILLOUT evaporationbarrier and brought to 95° C. for 10 minutes. The cleavage reactionswere then started by the addition of 5 μl of the desired enzyme/probemixture; reactions 1, 4 and 5 received CLEAVASE A/G nuclease. Reactions2 and 3 received CLEAVASE BN; reactions 6 and 7 received AmpliTaq®;reactions 8 and 9 received AmpliTaq® LD; and reactions 10 and 11received Taq DNA Polymerase from Fisher Scientific.

[0790] The reactions were incubated at 63° C. for 30 minutes and werestopped by the addition of 6 μl of 95% formamide with 20 mM EDTA and0.05% marker dyes. Samples were heated to 75° C. for 2 minutesimmediately before electrophoresis through a 20% acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA. The products of the reactions were visualized by the use ofan Hitachi FMBIO fluorescence imager, and the results are displayed inFIG. 33.

[0791] A pairwise comparison of the lanes shown in FIG. 33 without andwith the saliva extract, treated with each of the enzymes, shows thatthe saliva extract has different effects on each of the enzymes. Whilethe CLEAVASE BN nuclease and the AmpliTaq® are significantly inhibitedfrom cleaving in these conditions, the CLEAVASE A/G nuclease andAmpliTaq® LD display little difference in the yield of cleaved probe.The preparation of Taq DNA polymerase from Fisher Scientific shows anintermediate response, with a partial reduction in the yield of cleavedproduct. From the standpoint of polymerization, the three DNAPTaqvariants should be equivalent; these should be the same protein with thesame amount of synthetic activity. It is possible that the differencesobserved could be due to variations in the amount of nuclease activitypresent in each preparation caused by different handling duringpurification, or by different purification protocols. In any case,quality control assays designed to assess polymerization activity incommercial DNAP preparations would be unlikely to reveal variation inthe amount of nuclease activity present. If preparations of DNAPTaq werescreened for full 5′ nuclease activity (i.e., if the 5′ nucleaseactivity was specifically quantitated), it is likely that thepreparations would display sensitivities (to saliva extract) more inline with that observed using CLEAVASE A/G nuclease, from which DNAPTaqdiffers by a very few amino acids.

[0792] It is worthy of note that even in the slowed reactions ofCLEAVASE BN and the DNAPTaq variants there is no noticeable increase innon-specific cleavage of the probe oligonucleotide due to inappropriatehybridization or saliva-borne nucleases.

Example 16

[0793] Comparison of Additional 5′ Nucleases in the INVADER-DirectedCleavage Assay

[0794] A number of eubacterial Type A DNA polymerases (i.e., Pol I typeDNA polymerases) have been shown to function as structure specificendonucleases (See, Example 1, and Lyamichev et al., supra). In thisExample, it was demonstrated that the enzymes of this class can also bemade to catalyze the INVADER-directed cleavage of the present invention,albeit not as efficiently as the CLEAVASE enzymes.

[0795] CLEAVASE BN nuclease and CLEAVASE A/G nuclease were tested alongside three different thermostable DNA polymerases: Thermus aquaticus DNApolymerase (Promega), Thermus thermophilus and Thermus flavus DNApolymerases (Epicentre). The enzyme mixtures used in the reactions shownin lanes 1-11 of FIG. 34 contained the following, each in a volume of 5μl: Lane 1: 20 mM MOPS (pH 7.5) with 0.1% each of Tween 20 and NP-40, 4mM MnCl₂, 100 mM KCl; Lane 2: 25 ng of CLEAVASE BN nuclease in the samesolution described for lane 1; Lane 3: 2.25 μl of CLEAVASE A/G nucleaseextract (prepared as described in Example 2), in the same solutiondescribed for lane 1; Lane 4: 2.25 μl of CLEAVASE A/G nuclease extractin 20 mM Tris-Cl, (pH 8.5), 4 mM MgCl₂ and 100 mM KCl; Lane 5: 11.25polymerase units of Taq DNA polymerase in the same buffer described forlane 4; Lane 6: 11.25 polymerase units of Tth DNA polymerase in the samebuffer described for lane 1; Lane 7: 11.25 polymerase units of Tth DNApolymerase in a 2× concentration of the buffer supplied by themanufacturer, supplemented with 4 mM MnCl₂; Lane 8: 11.25 polymeraseunits of Tth DNA polymerase in a 2× concentration of the buffer suppliedby the manufacturer, supplemented with 4 mM MgCl₂; Lane 9: 2.25polymerase units of Tfl DNA polymerase in the same buffer described forlane 1; Lane 10: 2.25 polymerase units of Tfl polymerase in a 2×concentration of the buffer supplied by the manufacturer, supplementedwith 4 mM MnCl₂; Lane 11: 2.25 polymerase units of Tfl DNA polymerase ina 2× concentration of the buffer supplied by the manufacturer,supplemented with 4 mM MgCl₂.

[0796] Sufficient target DNA, probe and INVADER for all 11 reactions wascombined into a master mix. This mix contained 550 fmoles ofsingle-stranded M13mp19 target DNA, 550 pmoles of the INVADERoligonucleotide (SEQ ID NO: 35) and 55 pmoles of the probeoligonucleotide (SEQ ID NO: 32), each as depicted in FIG. 28c, in 55 μlof distilled water. Five μl of the DNA mixture was dispensed into eachof 11 labeled tubes and overlaid with a drop of CHILLOUT evaporationbarrier. The reactions were brought to 63° C. and cleavage was startedby the addition of 5 μl of the appropriate enzyme mixture. The reactionmixtures were then incubated at 63° C. temperature for 15 minutes. Thereactions were stopped by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. Samples were heated to 90° C. for 1minute immediately before electrophoresis through a 20% acrylamide gel(19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate (pH8.3), 1.4 mM EDTA. Following electrophoresis, the products of thereactions were visualized by the use of an Hitachi FMBIO fluorescenceimager, and the results are displayed in FIG. 34. Examination of theresults shown in FIG. 34 demonstrates that all of the 5′ nucleasestested have the ability to catalyze INVADER-directed cleavage in atleast one of the buffer systems tested. Although not optimized here,these cleavage agents are suitable for use in the methods of the presentinvention.

Example 17

[0797] The INVADER-Directed Cleavage Assay can Detect Single BaseDifferences in Target Nucleic Acid Sequences

[0798] The ability of the INVADER-directed cleavage assay to detectsingle base mismatch mutations was examined. Two target nucleic acidsequences containing CLEAVASE enzyme-resistant phosphorothioatebackbones were chemically synthesized and purified by polyacrylamide gelelectrophoresis. Targets comprising phosphorothioate backbones were usedto prevent exonucleolytic nibbling of the target when duplexed with anoligonucleotide. A target oligonucleotide, which provides a targetsequence that is completely complementary to the INVADER oligonucleotide(SEQ ID NO: 35) and the probe oligonucleotide (SEQ ID NO: 32), containedthe following sequence:

[0799] 5′-CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGC-3′ (SEQ ID NO: 36).A second target sequence containing a single base change relative to SEQID NO: 36 was synthesized: 5′-CCTTTCGCTCTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGC-3 (SEQ ID NO: 37; the single base change relative to SEQ IDNO: 36 is shown using bold and underlined type). The consequent mismatchoccurs within the “Z” region of the target as represented in FIG. 25.

[0800] To discriminate between two target sequences that differ by thepresence of a single mismatch), INVADER-directed cleavage reactions wereconducted using two different reaction temperatures (55° C. and 60° C).Mixtures containing 200 fmoles of either SEQ ID NO: 36 or SEQ ID NO: 37,3 pmoles of fluorescein-labeled probe oligonucleotide (SEQ ID NO: 32),7.7 pmoles of INVADER oligonucleotide (SEQ ID NO: 35) and 2 μl ofCLEAVASE A/G nuclease extract (prepared as described in Example 2) in 9μl of 10 mM MOPS (pH 7.4) with 50 mM KCl were assembled, covered with adrop of CHILLOUT evaporation barrier and brought to the appropriatereaction temperature. The cleavage reactions were initiated by theaddition of 1 μl of 20 mM MgCl₂. After 30 minutes at either 55° C. or60° C., 10 μl of 95% formamide with 20 mM EDTA and 0.05% marker dyes wasadded to stop the reactions. The reaction mixtures where then heated to90° C. for one minute prior to loading 4 μl onto 20% denaturingpolyacrylamide gels. The resolved reaction products were visualizedusing a Hitachi FMBIO fluorescence imager. The resulting image is shownin FIG. 35.

[0801] In FIG. 35, lanes 1 and 2 show the products from reactionsconducted at 55° C.; lanes 3 and 4 show the products from reactionsconducted at 60° C. Lanes 1 and 3 contained products from reactionscontaining SEQ ID NO: 36 (perfect match to probe) as the target. Lanes 2and 4 contained products from reactions containing SEQ ID NO: 37 (singlebase mis-match with probe) as the target. The target that does not havea perfect hybridization match (i.e., complete complementarity) with theprobe will not bind as strongly (i.e., the T_(m) of that duplex will belower than the T_(m) of the same region if perfectly matched). Theresults presented here show that reaction conditions can be varied toeither accommodate the mis-match (e.g., by lowering the temperature ofthe reaction) or to exclude the binding of the mismatched sequence(e.g., by raising the reaction temperature).

[0802] The results shown in FIG. 35 demonstrate that the specificcleavage event that occurs in INVADER-directed cleavage reactions can beeliminated by the presence of a single base mis-match between the probeoligonucleotide and the target sequence. Thus, reaction conditions canbe chosen so as to exclude the hybridization of mismatchedINVADER-directed cleavage probes thereby diminishing or even eliminatingthe cleavage of the probe. In an extension of this assay system,multiple cleavage probes, each possessing a separate reporter molecule(i.e., a unique label), could also be used in a single cleavagereaction, to simultaneously probe for two or more variants in the sametarget region. The products of such a reaction would allow not only thedetection of mutations that exist within a target molecule, but wouldalso allow a determination of the relative concentrations of eachsequence (i.e., mutant and wild type or multiple different mutants)present within samples containing a mixture of target sequences. Whenprovided in equal amounts, but in a vast excess (e.g., at least a100-fold molar excess; typically at least 1 pmole of each probeoligonucleotide would be used when the target sequence was present atabout 10 fmoles or less) over the target and used in optimizedconditions. As discussed above, any differences in the relative amountsof the target variants will not affect the kinetics of hybridization, sothe amounts of cleavage of each probe will reflect the relative amountsof each variant present in the reaction.

[0803] The results shown in the Example clearly demonstrate that theINVADER-directed cleavage reaction can be used to detect single basedifference between target nucleic acids.

Example 18

[0804] The INVADER-Directed Cleavage Reaction is Insensitive to LargeChanges in Reaction Conditions

[0805] The results shown above demonstrated that the INVADER-directedcleavage reaction can be used for the detection of target nucleic acidsequences and that this assay can be used to detect single basedifference between target nucleic acids. These results demonstrated that5′ nucleases (e.g., CLEAVASEBN, CLEAVASE A/G, DNAPTaq, DNAPTth, DNAPTfl)could be used in conjunction with a pair of overlapping oligonucleotidesas an efficient way to recognize nucleic acid targets. In theexperiments below it is demonstrated that invasive cleavage reaction isrelatively insensitive to large changes in conditions thereby making themethod suitable for practice in clinical laboratories.

[0806] The effects of varying the conditions of the cleavage reactionwere examined for their effect(s) on the specificity of the invasivecleavage and the on the amount of signal accumulated in the course ofthe reaction. To compare variations in the cleavage reaction a“standard” INVADER cleavage reaction was first defined. In eachinstance, unless specifically stated to be otherwise, the indicatedparameter of the reaction was varied, while the invariant aspects of aparticular test were those of this standard reaction. The results ofthese tests are either shown in FIGS. 38-40, or the results describedbelow.

[0807] a) The Standard INVADER-Directed Cleavage Reaction

[0808] The standard reaction was defined as comprising 1 fmole ofM13mp18 single-stranded target DNA (NEB), 5 pmoles of the labeled probeoligonucleotide (SEQ ID NO: 38), 10 pmole of the upstream INVADERoligonucleotide (SEQ ID NO: 39) and 2 units of CLEAVASE A/G in 10 μl of10 mM MOPS, pH 7.5 with 100 mM KCl, 4 mM MnCl₂, and 0.05% each Tween-20and Nonidet-P40. For each reaction, the buffers, salts and enzyme werecombined in a volume of 5 μl; the DNAs (target and two oligonucleotides)were combined in 5 μl of dH₂O and overlaid with a drop of CHILLOUTevaporation barrier. When multiple reactions were performed with thesame reaction constituents, these formulations were expandedproportionally.

[0809] Unless otherwise stated, the sample tubes with the DNA mixtureswere warmed to 61° C., and the reactions were started by the addition of5 μl of the enzyme mixture. After 20 minutes at this temperature, thereactions were stopped by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. Samples were heated to 75° C. for 2minutes immediately before electrophoresis through a 20% acrylamide gel(19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA. The products of the reactions were visualized by theuse of an Hitachi FMBIO fluorescence imager. In each case, the uncutprobe material was visible as an intense black band or blob, usually inthe top half of the panel, while the desired products of INVADERspecific cleavage were visible as one or two narrower black bands,usually in the bottom half of the panel. Under some reaction conditions,particularly those with elevated salt concentrations, a secondarycleavage product is also visible (thus generating a doublet). Ladders oflighter grey bands generally indicate either exonuclease nibbling of theprobe oligonucleotide or heat-induced, non-specific breakage of theprobe.

[0810]FIG. 37 depicts the annealing of the probe and INVADERoligonucleotides to regions along the M13mp18 target molecule (thebottom strand). In FIG. 37 only a 52 nucleotide portion of the M13mp18molecule is shown; this 52 nucleotide sequence is listed in SEQ ID NO:31 (this sequence is identical in both M13mp18 and M13mp19). The probeoligonucleotide (top strand) contains a Cy3 amidite label at the 5′ end;the sequence of the probe is 5′-AGAAAGGAAGGGAAGAAAGCGAAAGGT-3′ (SEQ IDNO: 38. The bold type indicates the presence of a modified base(2′-O—CH₃). Cy3 amidite (Pharmacia) is a indodicarbocyanine dye amiditethat can be incorporated at any position during the synthesis ofoligonucleotides; Cy3 fluoresces in the yellow region (excitation andemission maximum of 554 and 568 nm, respectively). The INVADERoligonucleotide (middle strand) has the following sequence:5′-GCCGGCGAACGTGGCGAGAAAGGA-3′. (SEQ ID NO:39)

[0811] b) KCl Titration

[0812]FIG. 38 shows the results of varying the KCl concentration incombination with the use of 2 mM MnCl₂, in an otherwise standardreaction. The reactions were performed in duplicate for confirmation ofobservations; the reactions shown in lanes 1 and 2 contained no addedKCl, lanes 3 and 4 contained KCl at 5 mM, lanes 5 and 6 contained 25 mMKCl, lanes 7 and 8 contained 50 mM KCl, lanes 9 and 10 contained 100 mMKCl and lanes 11 and 12 contained 200 mM KCl. These results show thatthe inclusion of KCl allows the generation of a specific cleavageproduct. While the strongest signal is observed at the 100 mM KClconcentration, the specificity of signal in the other reactions with KClat or above 25 mM indicates that concentrations in the full range (i.e.,25-200 mM) may be chosen if it is so desirable for any particularreaction conditions.

[0813] As shown in FIG. 38, the INVADER-directed cleavage reactionrequires the presence of salt (e.g., KCl) for effective cleavage tooccur. In other reactions, it has been found that KCl can inhibit theactivity of certain CLEAVASE enzymes when present at concentrationsabove about 25 mM. For example, in cleavage reactions using the S-60oligonucleotide shown in FIG. 26, in the absence of primer, the CLEAVASEBN enzyme loses approximately 50% of its activity in 50 mM KCl.Therefore, the use of alternative salts in the INVADER-directed cleavagereaction was examined. In these experiments, the potassium ion wasreplaced with either Na⁺ or Li⁺ or the chloride ion was replaced withglutamic acid. The replacement of KCl with alternative salts isdescribed below in Sections c-e.

[0814] c) NaCl Titration

[0815] NaCl was used in place of KCl at 75, 100, 150 or 200 mM, incombination with the use 2 mM MnCl₂, in an otherwise standard reaction.These results showed that NaCl can be used as a replacement for KCl inthe INVADER-directed cleavage reaction, with like concentration givinglike results, (i.e., the presence of NaCl, like KCl, enhances productaccumulation).

[0816] d) LiCl Titration

[0817] LiCl was used in place of KCl in otherwise standard reactions.Concentrations tested were 25, 50, 75, 100, 150 and 200 mM LiCl. Theresults demonstrated that LiCl can be used as a suitable replacement forKCl in the INVADER-directed cleavage reaction (i.e., the presence ofLiCl, like KCl, enhances product accumulation), in concentrations ofabout 100 mM or higher.

[0818] e) KGlu Titration p The results of using a glutamate salt ofpotassium (KGlu) in place of the more commonly used chloride salt (KCl)in reactions performed over a range of temperatures were examined. KGluhas been shown to be a highly effective salt source for some enzymaticreactions, showing a broader range of concentrations that permit maximumenzymatic activity (Leirmo et al., Biochem., 26:2095 [1987]). Theability of KGlu to facilitate the annealing of the probe and INVADERoligonucleotides to the target nucleic acid was compared to that ofLiCl. In these experiments, the reactions were run for 15 minutes,rather than the standard 20 minutes, in standard reactions that replacedKCl 200 mM, 300 mM or 400 mM KGlu. The reactions were run at 65° C., 67°C., 69° C. or 71° C. The results showed demonstrated that KGlu was veryeffective as a salt in the invasive cleavage reactions, with fullactivity apparent even at 400 mM KGlu, though at the lowest temperaturecleavage was reduced by about 30% at 300 mM KGlu, and by about 90% to400 mM KGlu.

[0819] i) MnCl₂ and MgCl₂ Titration and Ability to Replace MnCl₂ withMgCl₂

[0820] In some instances it may be desirable to perform the invasivecleavage reaction in the presence of Mg²⁺, either in addition to, or inplace of Mn²⁺ as the necessary divalent cation required for activity ofthe enzyme employed. For example, some common methods of preparing DNAfrom bacterial cultures or tissues use MgCl₂ in solutions that are usedto facilitate the collection of DNA by precipitation. In addition,elevated concentrations (i.e., greater than 5 mM) of divalent cation canbe used to facilitate hybridization of nucleic acids, in the same waythat the monovalent salts were used above, thereby enhancing theinvasive cleavage reaction. In this experiment, the tolerance of theinvasive cleavage reaction was examined for 1) the substitution of MgCl₂for MnCl₂ and for the ability to produce specific product in thepresence of increasing concentrations of MgCl₂ and MnCl₂.

[0821]FIG. 39 shows the results of either varying the concentration ofMnCl₂ from 2 mM to 8 mM, replacing the MnCl₂ with MgCl₂ at 2 to 4 mM, orof using these components in combination in an otherwise standardreaction. The reactions analyzed in lanes 1 and 2 contained 2 mM eachMnCl₂ and MgCl₂, lanes 3 and 4 contained 2 mM MnCl₂ only, lanes 5 and 6contained 3 mM MnCl₂, lanes 7 and 8 contained 4 mM MnCl₂, lanes 9 and 10contained 8 mM MnCl₂. The reactions analyzed in lanes 11 and 12contained 2 mM MgCl₂ and lanes 13 and 14 contained 4 mM MgCl₂. Theseresults show that both MnCl₂ and MgCl₂ can be used as the necessarydivalent cation to enable the cleavage activity of the CLEAVASE A/Genzyme in these reactions and that the invasive cleavage reaction cantolerate a broad range of concentrations of these components.

[0822] In addition to examining the effects of the salt environment onthe rate of product accumulation in the invasive cleavage reaction, theuse of reaction constituents shown to be effective in enhancing nucleicacid hybridization in either standard hybridization assays (e.g., blothybridization) or in ligation reactions was examined. These componentsmay act as volume excluders, increasing the effective concentration ofthe nucleic acids of interest and thereby enhancing hybridization, orthey may act as charge-shielding agents to minimize repulsion betweenthe highly charged backbones of the nucleic acids strands. The resultsof these experiments are described in Sections g and h below.

[0823] g) Effect of CTAB Addition

[0824] The polycationic detergent cetyltrietheylammonium bromide (CTAB)has been shown to dramatically enhance hybridization of nucleic acids(Pontius and Berg, Proc. Natl. Acad. Sci. USA 88:8237 [1991]). Theeffect of adding the detergent CTAB in concentrations from 100 mM to 1mM to invasive cleavage reactions in which 150 mM LiCl was used in placeof the KCl in otherwise standard reactions was also investigated. Theseresults showed that 200 mM CTAB may have a very moderate enhancingeffect under these reaction conditions, and the presence of CTAB inexcess of about 500 μM was inhibitory to the accumulation of specificcleavage product.

[0825] h) Effect of PEG Addition

[0826] The effect of adding polyethylene glycol (PEG) at 4.8 or 12%(w/v) concentrations to otherwise standard reactions was also examined.The effects of increasing the reaction temperature of the PEG-containingreactions was examined by performing duplicate sets of PEG titrationreactions at 61° C. and 65° C. The results showed that at allpercentages tested, and at both temperatures tested, the inclusion ofPEG substantially eliminated the production of specific cleavageproduct.

[0827] In addition to, the presence of 1× Denhardts in the reactionmixture was found to have no adverse effect upon the cleavage reaction(50× Denhardts contains per 500 ml: 5 g Ficoll, 5 gpolyvinylpyrrolidone, 5 g BSA). Further, the presence of each componentof Denhardt's was examined individually (i.e., Ficoll alone,polyvinylpyrrolidone alone, BSA alone) for the effect upon theINVADER-directed cleavage reaction; no adverse effect was observed.

[0828] i) Effect of the Addition of Stabilizing Agents

[0829] Another approach to enhancing the output of the invasive cleavagereaction is to enhance the activity of the enzyme employed, either byincreasing its stability in the reaction environment or by increasingits turnover rate. Without regard to the precise mechanism by whichvarious agents operate in the invasive cleavage reaction, a number ofagents commonly used to stabilize enzymes during prolonged storage weretested for the ability to enhance the accumulation of specific cleavageproduct in the invasive cleavage reaction.

[0830] The effects of adding glycerol at 15% and of adding thedetergents Tween-20 and Nonidet-P40 at 1.5%, alone or in combination, inotherwise standard reactions were also examined. The resultsdemonstrated that under these conditions these adducts had little or noeffect on the accumulation of specific cleavage product.

[0831] The effects of adding gelatin to reactions in which the saltidentity and concentration were varied from the standard reaction werealso investigated. The results demonstrated that in the absence of saltthe gelatin had a moderately enhancing effect on the accumulation ofspecific cleavage product, but when either salt (KCl or LiCl) was addedto reactions performed under these conditions, increasing amounts ofgelatin reduced the product accumulation.

[0832] j) Effect of Adding Large Amounts of Non-Target Nucleic Acid

[0833] In detecting specific nucleic acid sequences within samples, itis important to determine if the presence of additional genetic material(i.e., non-target nucleic acids) will have a negative effect on thespecificity of the assay. In this experiment, the effect of includinglarge amounts of non-target nucleic acid, either DNA or RNA, on thespecificity of the invasive cleavage reaction was examined. The data wasexamined for either an alteration in the expected site of cleavage, orfor an increase in the nonspecific degradation of the probeoligonucleotide.

[0834]FIG. 40 shows the effects of adding non-target nucleic acid (e.g.,genomic DNA or tRNA) to an invasive cleavage reaction performed at 65°C., with 150 mM LiCl in place of the KCl in the standard reaction. Thereactions assayed in lanes 1 and 2 contained 235 and 470 ng of genomicDNA, respectively. The reactions analyzed in lanes 3, 4, 5 and 6contained 100 ng, 200 ng, 500 ng and 1 μg of tRNA, respectively. Lane 7represents a control reaction that contained no added nucleic acidbeyond the amounts used in the standard reaction. The results shown inFIG. 40 demonstrate that the inclusion of non-target nucleic acid inlarge amounts could visibly slow the accumulation of specific cleavageproduct (while not limiting the invention to any particular mechanism,it is thought that the additional nucleic acid competes for binding ofthe enzyme with the specific reaction components). In additionalexperiments it was found that the effect of adding large amounts ofnon-target nucleic acid can be compensated for by increasing the enzymein the reaction. The data shown in FIG. 40 also demonstrate that a keyfeature of the invasive cleavage reaction, the specificity of thedetection, was not compromised by the presence of large amounts ofnon-target nucleic acid.

[0835] In addition to the data presented above, invasive cleavagereactions were run with succinate buffer at pH 5.9 in place of the MOPSbuffer used in the “standard” reaction; no adverse effects wereobserved.

[0836] The data shown in FIGS. 38-40 and described above demonstratethat the invasive cleavage reaction can be performed using a widevariety of reaction conditions and is therefore suitable for practice inclinical laboratories.

Example 19

[0837] Detection of RNA Targets by INVADER-Directed Cleavage

[0838] In addition to the clinical need to detect specific DNA sequencesfor infectious and genetic diseases, there is a need for technologiesthat can quantitatively detect target nucleic acids that are composed ofRNA. For example, a number of viral agents, such as hepatitis C virus(HCV) and human immunodeficiency virus (HIV) have RNA genomic material,the quantitative detection of which can be used as a measure of viralload in a patient sample. Such information can be of critical diagnosticor prognostic value.

[0839] Hepatitis C virus (HCV) infection is the predominant cause ofpost-transfusion non-A, non-B (NANB) hepatitis around the world. Inaddition, HCV is the major etiologic agent of hepatocellular carcinoma(HCC) and chronic liver disease world wide. The genome of HCV is a small(9.4 kb) RNA molecule. In studies of transmission of HCV by bloodtransfusion it has been found the presence of HCV antibody, as measuredin standard immunological tests, does not always correlate with theinfectivity of the sample, while the presence of HCV RNA in a bloodsample strongly correlates with infectivity. Conversely, serologicaltests may remain negative in immunosuppressed infected individuals,while HCV RNA may be easily detected (Cuthbert, Clin. Microbiol. Rev.,7:505 [1994]).

[0840] The need for and the value of developing a probe-based assay forthe detection the HCV RNA is clear. The polymerase chain reaction hasbeen used to detect HCV in clinical samples, but the problems associatedwith carry-over contamination of samples has been a concern. Directdetection of the viral RNA without the need to perform either reversetranscription or amplification would allow the elimination of several ofthe points at which existing assays may fail.

[0841] The genome of the positive-stranded RNA hepatitis C viruscomprises several regions including 5′ and 3′ noncoding regions (i.e.,5′ and 3′ untranslated regions) and a polyprotein coding region thatencodes the core protein (C), two envelope glycoproteins (E1 and E2/NS1)and six nonstructural glycoproteins (NS2-NS5b). Molecular biologicalanalysis of the HCV genome has showed that some regions of the genomeare very highly conserved between isolates, while other regions arefairly rapidly changeable. The 5′ noncoding region (NCR) is the mosthighly conserved region in the HCV. These analyses have allowed theseviruses to be divided into six basic genotype groups, and then furtherclassified into over a dozen sub-types (the nomenclature and division ofHCV genotypes is evolving; see Altamirano et al., J. Infect. Dis.,171:1034 (1995) for a recent classification scheme).

[0842] In order to develop a rapid and accurate method of detecting HCVpresent in infected individuals, the ability of the INVADER-directedcleavage reaction to detect HCV RNA was examined. Plasmids containingDNA derived from the conserved 5′-untranslated region of six differentHCV RNA isolates were used to generate templates for in vitrotranscription. The HCV sequences contained within these six plasmidsrepresent genotypes 1 (four sub-types represented; 1a, 1b, 1c, and Δ1c),2, and 3. The nomenclature of the HCV genotypes used herein is that ofSimmonds et al. (as described in Altamirano et al., supra). The Δ1csubtype was used in the model detection reaction described below.

[0843] a) Generation of Plasmids Containing HCV Sequences

[0844] Six DNA fragments derived from HCV were generated by RT-PCR usingRNA extracted from serum samples of blood donors; these PCR fragmentswere a gift of Dr. M. Altamirano (University of British Columbia,Vancouver). These PCR fragments represent HCV sequences derived from HCVgenotypes 1a, 1b, 1c, Δ1c, 2c and 3a.

[0845] The RNA extraction, reverse transcription and PCR were performedusing standard techniques (Altamirano et al., supra). Briefly, RNA wasextracted from 100 μl of serum using guanidine isothiocyanate, sodiumlauryl sarkosate and phenol-chloroform (Inchauspe et al., Hepatol.,14:595 [1991]). Reverse transcription was performed according to themanufacturer's instructions using a GeneAmp rTh reverse transcriptaseRNA PCR kit (Perkin-Elmer) in the presence of an external antisenseprimer, HCV342. The sequence of the HCV342 primer is5′-GGTTTTTCTTTGAGGTTTAG-3′ (SEQ ID NO: 40). Following termination of theRT reaction, the sense primer HCV7 (5′-GCGACACTCCACCATAGAT-3′ [SEQ IDNO: 41]) and magnesium were added and a first PCR was performed.Aliquots of the first PCR products were used in a second (nested) PCR inthe presence of primers HCV46 (5′-CTGTCTTCACGCAGAAAGC-3′ [SEQ ID NO:42]) and HCV308 [5′-GCACGGT CTACGAGACCTC-3′ [SEQ ID NO: 43]). The PCRsproduced a 281 bp product that corresponds to a conserved 5′ noncodingregion (NCR) region of HCV between positions −284 and −4 of the HCVgenome (Altramirano et al., supra).

[0846] The six 281 bp PCR fragments were used directly for cloning orthey were subjected to an additional amplification step using a 50 μlPCR comprising approximately 100 fmoles of DNA, the HCV46 and HCV308primers at 0.1 μM, 100 μM of all four dNTPs and 2.5 units of Taq DNApolymerase in a buffer containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5mM MgCl₂ and 0.1% Tween 20. The PCRs were cycled 25 times at 96° C. for45 sec., 55° C. for 45 sec. and 72° C. for 1 min. Two microliters ofeither the original DNA samples or the reamplified PCR products wereused for cloning in the linear pT7Blue T-vector (Novagen) according tomanufacturer's protocol. After the PCR products were ligated to thepT7Blue T-vector, the ligation reaction mixture was used to transformcompetent JM109 cells (Promega). Clones containing the pT7Blue T-vectorwith an insert were selected by the presence of colonies having a whitecolor on LB plates containing 40 μg/ml X-Gal, 40 μg/ml IPTG and 50 μg/mlampicillin. Four colonies for each PCR sample were picked and grownovernight in 2 ml LB media containing 50 μg/ml carbenicillin. PlasmidDNA was isolated using the following alkaline miniprep protocol. Cellsfrom 1.5 ml of the overnight culture were collected by centrifugationfor 2 min. in a microcentrifuge (14K rpm), the supernatant was discardedand the cell pellet was resuspended in 50 μl TE buffer with 10 μg/mlRNAse A (Pharmacia). One hundred microliters of a solution containing0.2 N NaOH, 1% SDS was added and the cells were lysed for 2 min. Thelysate was gently mixed with 100 μl of 1.32 M potassium acetate, pH 4.8,and the mixture was centrifuged for 4 min. in a microcentrifuge (14Krpm); the pellet comprising cell debris was discarded. Plasmid DNA wasprecipitated from the supernatant with 200 μl ethanol and pelleted bycentrifugation a microcentrifuge (14K rpm). The DNA pellet was air driedfor 15 min. and was then redissolved in 50 μl TE buffer (10 mM Tris-HCl,pH 7.8, 1 mM EDTA).

[0847] b) Reamplifilcation of HCV Clones to Add the Phage T7 Promoterfor Subsequent in Vitro Transcription

[0848] To ensure that the RNA product of transcription had a discrete 3′end it was necessary to create linear transcription templates thatstopped at the end of the HCV sequence. These fragments wereconveniently produced using the PCR to reamplify the segment of theplasmid containing the phage promoter sequence and the HCV insert. Forthese studies, the clone of HCV type Δ1c was reamplified using a primerthat hybridizes to the T7 promoter sequence: 5′-TAATACGACTCACTATAGGG-3′(SEQ ID NO: 44; “the T7 promoter primer”) (Novagen) in combination withthe 3′ terminal HCV-specific primer HCV308 (SEQ ID NO: 43). For thesereactions, 1 μl of plasmid DNA (approximately 10 to 100 ng) wasreamplified in a 200 μl PCR using the T7 and HCV308 primers as describedabove with the exception that 30 cycles of amplification were employed.The resulting amplicon was 354 bp in length. After amplification the PCRmixture was transferred to a fresh 1.5 ml microcentrifuge tube, themixture was brought to a final concentration of 2 M NH₄OAc, and theproducts were precipitated by the addition of one volume of 100%isopropanol. Following a 10 min. incubation at room temperature, theprecipitates were collected by centrifugation, washed once with 80%ethanol and dried under vacuum. The collected material was dissolved in100 μl nuclease-free distilled water (Promega).

[0849] Segments of RNA were produced from this amplicon by in vitrotranscription using the RiboMAX™ Large Scale RNA Production System(Promega) in accordance with the manufacturer's instructions, using 5.3μg of the amplicon described above in a 100 μl reaction. Thetranscription reaction was incubated for 3.75 hours, after which the DNAtemplate was destroyed by the addition of 5-6 μl of RQ1 RNAse-free DNAse(1 unit/μl) according to the RiboMAX™ kit instructions. The reaction wasextracted twice with phenol/chloroform/isoamyl alcohol (50:48:2) and theaqueous phase was transferred to a fresh microcentrifuge tube. The RNAwas then collected by the addition of 10 μl of 3M NH₄OAc, pH 5.2 and 110μl of 100% isopropanol. Following a 5 min. incubation at 4° C., theprecipitate was collected by centrifugation, washed once with 80%ethanol and dried under vacuum. The sequence of the resulting RNAtranscript (HCV1.1 transcript) is listed in SEQ ID NO: 45.

[0850] c) Detection of the HCV1.1 Transcript in the INVADER-DirectedCleavage Assay

[0851] Detection of the HCV1.1 transcript was tested in theINVADER-directed cleavage assay using an HCV-specific probeoligonucleotide (5′-CCGGTCGTCCTGGCAAT XCC-3′ [SEQ ID NO: 46]); Xindicates the presence of a fluorescein dye on an abasic linker) and anHCV-specific INVADER oligonucleotide (5′-GTTTATCCAAGAAAGGAC CCGGTC-3′[SEQ ID NO: 47]) that causes a 6-nucleotide invasive cleavage of theprobe.

[0852] Each 10 μl of reaction mixture comprised 5 pmole of the probeoligonucleotide (SEQ ID NO: 46) and 10 pmole of the INVADERoligonucleotide (SEQ ID NO: 47) in a buffer of 10 mM MOPS, pH 7.5 with50 mM KCl, 4 mM MnCl₂, 0.05% each Tween-20 and Nonidet-P40 and 7.8 unitsRNasin® ribonuclease inhibitor (Promega). The cleavage agents employedwere CLEAVASE A/G (used at 5.3 ng/10 μl reaction) or DNAPTth (used at 5polymerase units/10 μl reaction). The amount of RNA target was varied asindicated below. When RNAse treatment is indicated, the target RNAs werepre-treated with 10 μg of RNase A (Sigma) at 37° C. for 30 min. todemonstrate that the detection was specific for the RNA in the reactionand not due to the presence of any residual DNA template from thetranscription reaction. RNase-treated aliquots of the HCV RNA were useddirectly without intervening purification.

[0853] For each reaction, the target RNAs were suspended in the reactionsolutions as described above, but lacking the cleavage agent and theMnCl₂ for a final volume of 10 μl, with the INVADER and probe at theconcentrations listed above. The reactions were warmed to 46° C. and thereactions were started by the addition of a mixture of the appropriateenzyme with MnCl₂. After incubation for 30 min. at 46° C., the reactionswere stopped by the addition of 8 μl of 95% formamide, 10 mM EDTA and0.02% methyl violet (methyl violet loading buffer). Samples were thenresolved by electrophoresis through a 15% denaturing polyacrylamide gel(19:1 cross-linked), containing 7 M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. Following electrophoresis, the labeledreaction products were visualized using the FMBIO-100 Image Analyzer(Hitachi), with the resulting imager scan shown in FIG. 41.

[0854] In FIG. 41, the samples analyzed in lanes 1-4 contained 1 pmoleof the RNA target, the reactions shown in lanes 5-8 contained 100 fmolesof the RNA target and the reactions shown in lanes 9-12 contained 10fmoles of the RNA target. All odd-numbered lanes depict reactionsperformed using CLEAVASE A/G enzyme and all even-numbered lanes depictreactions performed using DNAPTth. The reactions analyzed in lanes 1, 2,5, 6, 9 and 10 contained RNA that had been pre-digested with RNase A.These data demonstrate that the invasive cleavage reaction efficientlydetects RNA targets and further, the absence of any specific cleavagesignal in the RNase-treated samples confirms that the specific cleavageproduct seen in the other lanes is dependent upon the presence of inputRNA.

Example 20

[0855] The Fate of the Target RNA in the INVADER-Directed CleavageReaction

[0856] In this Example, the fate of the RNA target in theINVADER-directed cleavage reaction was examined. As shown above inExample ID, when RNAs are hybridized to DNA oligonucleotides, the 5′nucleases associated with DNA polymerases can be used to cleave theRNAs; such cleavage can be suppressed when the 5′ arm is long or when itis highly structured (Lyamichev et al., Science 260:778 [1993], and U.S.Pat. No. 5,422,253, the disclosure of which is herein incorporated byreference). In this experiment, the extent to which the RNA target wouldbe cleaved by the cleavage agents when hybridized to the detectionoligonucleotides (i.e., the probe and INVADER oligonucleotides) wasexamined using reactions similar to those described in Example 20,performed using fluorescein-labeled RNA as a target.

[0857] Transcription reactions were performed as described in Example 19with the exception that 2% of the UTP in the reaction was replaced withfluorescein-12-UTP (Boehringer Mannheim) and 5.3 μg of the amplicon wasused in a 100 μl reaction. The transcription reaction was incubated for2.5 hours, after which the DNA template was destroyed by the addition of5-6 μg of RQ1 RNAse-free DNAse (1 unit/μl) according to the RiboMAX™ kitinstructions. The organic extraction was omitted and the RNA wascollected by the addition of 10 μl of 3M NaOAc, pH 5.2 and 110 μl of100% isopropanol. Following a 5 min. incubation at 4° C., theprecipitate was collected by centrifugation, washed once with 80%ethanol and dried under vacuum. The resulting RNA was dissolved in 100μl of nuclease-free water. Half (i.e., 50%) of the sample was purifiedby electrophoresis through a 8% denaturing polyacrylamide gel (19:1cross-linked), containing 7 M urea, in a buffer of 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA. The gel slice containing the full-length material wasexcised and the RNA was eluted by soaking the slice overnight at 4° C.in 200 μl of 10 mM Tris-Cl, pH 8.0, 0.1 mM EDTA and 0.3 M NaOAc. The RNAwas then precipitated by the addition of 2.5 volumes of 100% ethanol.After incubation at −20° C. for 30 min., the precipitates were recoveredby centrifugation, washed once with 80% ethanol and dried under vacuum.The RNA was dissolved in 25 μg of nuclease-free water and thenquantitated by UV absorbance at 260 nm.

[0858] Samples of the purified RNA target were incubated for 5 or 30min. in reactions that duplicated the CLEAVASE A/G and DNAPTth INVADERreactions described in Example 20 with the exception that the reactionslacked probe and INVADER oligonucleotides. Subsequent analysis of theproducts showed that the RNA was very stable, with a very slightbackground of non-specific degradation, appearing as a gray backgroundin the gel lane. The background was not dependent on the presence ofenzyme in the reaction.

[0859] INVADER detection reactions using the purified RNA target wereperformed using the probe/INVADER pair described in Example 19 (SEQ IDNOS: 46 and 47). Each reaction included 500 fmole of the target RNA, 5pmoles of the fluorescein-labeled probe and 10 pmoles of the INVADERoligonucleotide in a buffer of 10 nM MOPS, pH 7.5 with 150 mM LiCl, 4 mMMnCl₂, 0.05% each Tween-20 and Nonidet-P40 and 39 units RNAsin®(Promega). These components were combined and warmed to 50° C. and thereactions were started by the addition of either 53 ng of CLEAVASE A/Gor 5 polymerase units of DNAPTth. The final reaction volume was 10 μl.After 5 min at 50° C., 5 μl aliquots of each reaction were removed totubes containing 4 μl of 95% formamide, 10 mM EDTA and 0.02% methylviolet. The remaining aliquot received a drop of CHILLOUT evaporationbarrier and was incubated for an additional 25 min. These reactions werethen stopped by the addition of 4 μl of the above formamide solution.The products of these reactions were resolved by electrophoresis throughseparate 20% denaturing polyacrylamide gels (19:1 cross-linked),containing 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mMEDTA. Following electrophoresis, the labeled reaction products werevisualized using the FMBIO-100 Image Analyzer (Hitachi), with theresulting imager scans shown in FIGS. 42A (5 min reactions) and 42B (30min. reactions).

[0860] In FIG. 53 the target RNA is seen very near the top of each lane,while the labeled probe and its cleavage products are seen just belowthe middle of each panel. The FMBIO-100 Image Analyzer was used toquantitate the fluorescence signal in the probe bands. In each panel,lane 1 contains products from reactions performed in the absence of acleavage agent, lane 2 contains products from reactions performed usingCLEAVASE A/G and lane 3 contains products from reactions performed usingDNAPTth.

[0861] Quantitation of the fluorescence signal in the probe bandsrevealed that after a 5 min. incubation, 12% or 300 fmole of the probewas cleaved by the CLEAVASE A/G and 29% or 700 fmole was cleaved by theDNAPTth. After a 30 min. incubation, CLEAVASE A/G had cleaved 32% of theprobe molecules and DNAPTth had cleaved 70% of the probe molecules. (Theimages shown in FIGS. 42A and 42B were printed with the intensityadjusted to show the small amount of background from the RNAdegradation, so the bands containing strong signals are saturated andtherefore these images do not accurately reflect the differences inmeasured fluorescence)

[0862] The data shown in FIG. 42 clearly shows that, under invasivecleavage conditions, RNA molecules are sufficiently stable to bedetected as a target and that each RNA molecule can support many roundsof probe cleavage.

Example 21

[0863] Titration of Target RNA in the INVADER-Directed Cleavage Assay

[0864] One of the primary benefits of the INVADER-directed cleavageassay as a means for detection of the presence of specific targetnucleic acids is the correlation between the amount of cleavage productgenerated in a set amount of time and the quantity of the nucleic acidof interest present in the reaction. The benefits of quantitativedetection of RNA sequences was discussed in Example 19. In this Example,the quantitative nature of the detection assay was demonstrated throughthe use of various amounts of target starting material. In addition todemonstrating the correlation between the amounts of input target andoutput cleavage product, these data graphically show the degree to whichthe RNA target can be recycled in this assay.

[0865] The RNA target used in these reactions was thefluorescein-labeled material described in Example 20 (i.e., SEQ ID NO:45). Because the efficiency of incorporation of the fluorescein-12-UTPby the T7 RNA polymerase was not known, the concentration of the RNA wasdetermined by measurement of absorbance at 260 nm, not by fluorescenceintensity. Each reaction comprised 5 pmoles of the fluorescein-labeledprobe (SEQ ID NO: 46) and 10 pmoles of the INVADER oligonucleotide (SEQID NO: 47) in a buffer of 10 mM MOPS, pH 7.5 with 150 mM LiCl, 4 mMMnCl₂, 0.05% each Tween-20 and Nonidet-P40 and 39 units of RNAsin®(Promega). The amount of target RNA was varied from 1 to 100 fmoles, asindicated below. These components were combined, overlaid with CHILLOUTevaporation barrier and warmed to 50° C.; the reactions were started bythe addition of either 53 ng of CLEAVASE A/G or 5 polymerase units ofDNAPTth, to a final reaction volume of 10 μl. After 30 minutes at 50°C., reactions were stopped by the addition of 8 μl of 95% formamide, 10mM EDTA and 0.02% methyl violet. The unreacted markers in lanes 1 and 2were diluted in the same total volume (18 μl). The samples were heatedto 90° C. for 1 minute and 2.5 μl of each of these reactions wereresolved by electrophoresis through a 20% denaturing polyacrylamide gel(19:1 cross link) with 7M urea in a buffer of 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA, and the labeled reaction products were visualized using theFMBIO-100 Image Analyzer (Hitachi), with the resulting imager scansshown in FIG. 43.

[0866] In FIG. 43, lanes 1 and 2 show 5 pmoles of uncut probe and 500fmoles of untreated RNA, respectively. The probe is the very dark signalnear the middle of the panel, while the RNA is the thin line near thetop of the panel. These RNAs were transcribed with a 2% substitution offluorescein-12-UTP for natural UTP in the transcription reaction. Theresulting transcript contains 74 U residues, which would give an averageof 1.5 fluorescein labels per molecule. With one tenth the molar amountof RNA loaded in lane 2, the signal in lane 2 should be approximatelyone seventh (0.1 5×) the fluorescence intensity of the probe in lane 1.Measurements indicated that the intensity was closer to one fortieth,indicating an efficiency of label incorporation of approximately 17%.Because the RNA concentration was verified by A260 measurement this doesnot alter the experimental observations below, but it should be notedthat the signal from the RNA and the probes does not accurately reflectthe relative amounts in the reactions.

[0867] The reactions analyzed in lanes 3 through 7 contained 1, 5, 10,50 and 100 fmoles of target, respectively, with cleavage of the probeaccomplished by CLEAVASE A/G. The reactions analyzed in lanes 8 through12 repeated the same array of target amounts, with cleavage of the probeaccomplished by DNAPTth. The boxes seen surrounding the product bandsshow the area of the scan in which the fluorescence was measured foreach reaction. The number of fluorescence units detected within each boxis indicated below each box; background florescence was also measured.

[0868] It can be seen by comparing the detected fluorescence in eachlane that the amount of product formed in these 30 minute reactions canbe correlated to the amount of target material. The accumulation ofproduct under these conditions is slightly enhanced when DNAPTth is usedas the cleavage agent, but the correlation with the amount of targetpresent remains. This demonstrates that the INVADER assay can be used asa means of measuring the amount of target RNA within a sample.

[0869] Comparison of the fluorescence intensity of the input RNA withthat of the cleaved product shows that the INVADER-directed cleavageassay creates signal in excess of the amount of target, so that thesignal visible as cleaved probe is far more intense than thatrepresenting the target RNA. This further confirms the results describedin Example 20, in which it was demonstrated that each RNA molecule couldbe used many times.

Example 22

[0870] Detection of DNA by Charge Reversal

[0871] The detection of specific targets is achieved in theINVADER-directed cleavage assay by the cleavage of the probeoligonucleotide. In addition to the methods described in the precedingExamples, the cleaved probe may be separated from the uncleaved probeusing the charge reversal technique described below. This novelseparation technique is related to the observation that positivelycharged adducts can affect the electrophoretic behavior of smalloligonucleotides because the charge of the adduct is significantrelative to charge of the whole complex. Observations of aberrantmobility due to charged adducts have been reported in the literature,but in all cases found, the applications pursued by other scientistshave involved making oligonucleotides larger by enzymatic extension. Asthe negatively charged nucleotides are added on, the positive influenceof the adduct is reduced to insignificance. As a result, the effects ofpositively charged adducts have been dismissed and have receivedinfinitesimal notice in the existing literature.

[0872] This observed effect is of particular utility in assays based onthe cleavage of DNA molecules. When an oligonucleotide is shortenedthrough the action of a CLEAVASE enzyme or other cleavage agent, thepositive charge can be made to not only significantly reduce the netnegative charge, but to actually override it, effectively “flipping” thenet charge of the labeled entity. This reversal of charge allows theproducts of target-specific cleavage to be partitioned from uncleavedprobe by extremely simple means. For example, the products of cleavagecan be made to migrate towards a negative electrode placed at any pointin a reaction vessel, for focused detection without gel-basedelectrophoresis. When a slab gel is used, sample wells can be positionedin the center of the gel, so that the cleaved and uncleaved probes canbe observed to migrate in opposite directions. Alternatively, atraditional vertical gel can be used, but with the electrodes reversedrelative to usual DNA gels (i.e., the positive electrode at the top andthe negative electrode at the bottom) so that the cleaved moleculesenter the gel, while the uncleaved disperse into the upper reservoir ofelectrophoresis buffer.

[0873] An additional benefit of this type of readout is that theabsolute nature of the partition of products from substrates means thatan abundance of uncleaved probe can be supplied to drive thehybridization step of the probe-based assay, yet the unconsumed probecan be subtracted from the result to reduce background.

[0874] Through the use of multiple positively charged adducts, syntheticmolecules can be constructed with sufficient modification that thenormally negatively charged strand is made nearly neutral. When soconstructed, the presence or absence of a single phosphate group canmean the difference between a net negative or a net positive charge.This observation has particular utility when one objective is todiscriminate between enzymatically generated fragments of DNA, whichlack a 3′ phosphate, and the products of thermal degradation, whichretain a 3′ phosphate (and thus two additional negative charges).

[0875] a) Characterization of the Products of Thermal Breakage of DNAOligonucleotides

[0876] Thermal degradation of DNA probes results in high background thatcan obscure signals generated by specific enzymatic cleavage, decreasingthe signal-to-noise ratio. To better understand the nature of DNAthermal degradation products, the 5′ tetrachloro-fluorescein(TET)-labeled oligonucleotides 78 (SEQ ID NO: 48) and 79 (SEQ ID NO: 49)(100 pmole each) were incubated in 50 μl 10 mM NaCO₃ (pH 10.6), 50 mMNaCl at 90° C. for 4 hours. To prevent evaporation of the samples, thereaction mixture was overlaid with 50 μl of CHILLOUT liquid wax. Thereactions were then divided in two equal aliquots (A and B). Aliquot Awas mixed with 25 μl of methyl violet loading buffer and Aliquot B wasdephosphorylated by addition of 2.5 μl of 100 mM MgCl₂ and 1 μl of 1unit/μl Calf Intestinal Alkaline Phosphatase (CIAP) (Promega), withincubation at 37° C. for 30 min. after which 25 μl of methyl violetloading buffer was added. One microliter of each sample was resolved byelectrophoresis through a 12% polyacrylamide denaturing gel and imagedas described in Example 21; a 585 nm filter was used with the FMBIOImage Analyzer. The resulting imager scan is shown in FIG. 44.

[0877] In FIG. 44, lanes 1-3 contain the TET-labeled oligonucleotide 78and lanes 4-6 contain the TET-labeled oligonucleotides 79. Lanes 1 and 4contain products of reactions that were not heat treated. Lanes 2 and 5contain products from reactions that were heat treated and lanes 3 and 6contain products from reactions that were heat treated and subjected tophosphatase treatment.

[0878] As shown in FIG. 44, heat treatment causes significant breakdownof the 5′-TET-labeled DNA, generating a ladder of degradation products(FIG. 44, lanes 2, 3, 5 and 6). Band intensities correlate with purineand pyrimidine base positioning in the oligonucleotide sequences,indicating that backbone hydrolysis may occur through formation ofabasic intermediate products that have faster rates for purines than forpyrimidines (Lindahl and Karlström, Biochem., 12:5151 [1973]).

[0879] Dephosphorylation decreases the mobility of all productsgenerated by the thermal degradation process, with the most pronouncedeffect observed for the shorter products (FIG. 44, lanes 3 and 6). Thisdemonstrates that thermally degraded products possess a 3′ end terminalphosphoryl group that can be removed by dephosphorylation with CIAP.Removal of the phosphoryl group decreases the overall negative charge by2. Therefore, shorter products that have a small number of negativecharges are influenced to a greater degree upon the removal of twocharges. This leads to a larger mobility shift in the shorter productsthan that observed for the larger species.

[0880] The fact that the majority of thermally degraded DNA productscontain 3′ end phosphate groups and CLEAVASE enzyme-generated productsdo not allowed the development of simple isolation methods for productsgenerated in the INVADER-directed cleavage assay. The extra two chargesfound in thermal breakdown products do not exist in the specificcleavage products. Therefore, if one designs assays that producespecific products that contain a net positive charge of one or two, thensimilar thermal breakdown products will either be negative or neutral.The difference can be used to isolate specific products by reversecharge methods as shown below.

[0881] b) Dephosphorylation of Short Amino-Modified Oligonucleotides canReverse the Net Charge of the Labeled Product

[0882] To demonstrate how oligonucleotides can be transformed from netnegative to net positively charged compounds, the four shortamino-modified oligonucleotides labeled 70, 74, 75 and 76 and shown inFIGS. 45-47 were synthesized (FIG. 45 shows both oligonucleotides 70 and74). All four modified oligonucleotides possess Cy-3 dyes positioned atthe 5′-end, which individually are positively charged under reaction andisolation conditions described in this Example. Compounds 70 and 74contain two amino modified thymidines that, under reaction conditions,display positively charged R—NH₃ ⁺ groups attached at the C5 positionthrough a C₁₀ or C₆ linker, respectively. Because compounds 70 and 74are 3′-end phosphorylated, they consist of four negative charges andthree positive charges. Compound 75 differs from 74 in that the internalC₆ amino modified thymidine phosphate in 74 is replaced by a thymidinemethyl phosphonate. The phosphonate backbone is uncharged and so thereare a total of three negative charges on compound 75. This givescompound 75 a net negative one charge. Compound 76 differs from 70 inthat the internal amino modified thymidine is replaced by an internalcytosine phosphonate. The pK_(a) of the N3 nitrogen of cytosine can befrom 4 to 7. Thus, the net charges of this compound, can be from −1 to 0depending on the pH of the solution. For the simplicity of analysis,each group is assigned a whole number of charges, although it isrealized that, depending on the pK_(a) of each chemical group andambient pH, a real charge may differ from the whole number assigned. Itis assumed that this difference is not significant over the range of pHsused in the enzymatic reactions studied here.

[0883] Dephosphorylation of these compounds, or the removal of the 3′end terminal phosphoryl group, results in elimination of two negativecharges and generates products that have a net positive charge of one.In this experiment, the method of isoelectric focusing (IEF) was used todemonstrate a change from one negative to one positive net charge forthe described substrates during dephosphorylation.

[0884] Substrates 70, 74, 75 and 76 were synthesized by standardphosphoramidite chemistries and deprotected for 24 hours at 22° C. in 14M aqueous ammonium hydroxide solution, after which the solvent wasremoved in vacuo. The dried powders were resuspended in 200 μl of H₂Oand filtered through 0.2 μm filters. The concentration of the stocksolutions was estimated by UV-absorbance at 261 nm of samples diluted200-fold in H₂O using a spectrophotometer (Spectronic Genesys 2, MiltonRoy, Rochester, N.Y.).

[0885] Dephosphorylation of compounds 70 and 74, 75 and 76 wasaccomplished by treating 10 μl of the crude stock solutions (ranging inconcentration from approximately 0.5 to 2 mM) with 2 units of CIAP in100 μl of CIAP buffer (Promega) at 37° C. for 1 hour. The reactions werethen heated to 75° C. for 15 min. in order to inactivate the CLAP. Forclarity, dephosphorylated compounds are designated ‘dp’. For example,after dephosphorylation, substrate 70 becomes 70dp.

[0886] To prepare samples for IEF experiments, the concentration of thestock solutions of substrate and dephosphorylated product were adjustedto a uniform absorbance of 8.5×10⁻³ at 532 nm by dilution with water.Two microliters of each sample were analyzed by IEF using a PhastSystemelectrophoresis unit (Pharmacia) and PhastGel IEF 3-9 media (Pharmacia)according to the manufacturer's protocol. Separation was performed at15° C. with the following program: pre-run; 2,000 V, 2.5 mA, 3.5 W, 75Vh; load; 200 V, 2.5 mA, 3.5 W, 15 Vh; run; 2,000 V; 2.5 mA; 3.5 W, 130Vh. After separation, samples were visualized by using the FMBIO ImageAnalyzer (Hitachi) fitted with a 585 nm filter. The resulting imagerscan is shown in FIG. 48.

[0887]FIG. 48 shows results of IEF separation of substrates 70, 74, 75and 76 and their dephosphorylated products. The arrow labeled “SampleLoading Position” indicates a loading line, the ‘+’ sign shows theposition of the positive electrode and the ‘−’ sign indicates theposition of the negative electrode.

[0888] The results shown in FIG. 48 demonstrate that substrates 70, 74,75 and 76 migrated toward the positive electrode, while thedephosphorylated products 70dp, 74dp, 75dp and 76dp migrated towardnegative electrode. The observed differences in mobility direction wasin accord with predicted net charge of the substrates (minus one) andthe products (plus one). Small perturbations in the mobilities of thephosphorylated compounds indicate that the overall pIs vary. This wasalso true for the dephosphorylated compounds. The presence of thecytosine in 76dp, for instance, moved this compound further toward thenegative electrode, which was indicative of a higher overall pI relativeto the other dephosphorylated compounds. It is important to note thatadditional positive charges can be obtained by using a combination ofnatural amino modified bases (70dp and 74dp) along with unchargedmethylphosphonate bridges (products 75dp and 76dp).

[0889] The results shown above demonstrate that the removal of a singlephosphate group can flip the net charge of an oligonucleotide to causereversal in an electric field, allowing easy separation of products, andthat the precise base composition of the oligonucleotides affectabsolute mobility but not the charge-flipping effect.

Example 23

[0890] Detection of Specific Cleavage Products in the INVADER-DirectedCleavage Reaction by Charge Reversal

[0891] In this Example the ability to isolate products generated in theINVADER-directed cleavage assay from all other nucleic acids present inthe reaction cocktail was demonstrated using charge reversal. Thisexperiment utilized the following Cy3-labeled oligonucleotide:5′-Cy3-AminoT-AminoT-CTTTTCACCAGCGAGACGGG-3′ (SEQ ID NO: 50; termed“oligo 61”). Oligo 61 was designed to release upon cleavage a netpositively charged labeled product. To test whether or not a netpositively charged 5′-end labeled product would be recognized by theCLEAVASE enzymes in the INVADER-directed cleavage assay format, probeoligo 61 (SEQ ID NO: 50) and invading oligonucleotide 67 (SEQ ID NO: 51)were chemically synthesized on a DNA synthesizer (ABI 391) usingstandard phosphoramidite chemistries and reagents obtained from GlenResearch (Sterling, Va.).

[0892] Each assay reaction comprised 100 fmoles of M13mp18 singlestranded DNA, 10 pmoles each of the probe (SEQ ID NO: 50) and INVADER(SEQ ID NO: 51) oligonucleotides, and 20 units of CLEAVASE A/G in a 10μl solution of 10 mM MOPS, pH 7.4 with 100 mM KCl. Samples were overlaidwith mineral oil to prevent evaporation. The samples were brought toeither 50° C., 55° C., 60° C., or 65° C. and cleavage was initiated bythe addition of 1 μl of 40 mM MnCl₂. Reactions were allowed to proceedfor 25 minutes and then were terminated by the addition of 10 μl of 95%formamide containing 20 mM EDTA and 0.02% methyl violet. The negativecontrol experiment lacked the target M13mp18 and was run at 60° C. Fivemicroliters of each reaction were loaded into separate wells of a 20%denaturing polyacrylamide gel (cross-linked 29:1) with 8 M urea in abuffer containing 45 mM Tris-Borate (pH 8.3) and 1.4 mM EDTA. Anelectric field of 20 watts was applied for 30 minutes, with theelectrodes oriented as indicated in FIG. 49B (i.e., in reverseorientation). The products of these reactions were visualized using theFMBIO fluorescence imager and the resulting imager scan is shown in FIG.49B.

[0893]FIG. 49A provides a schematic illustration showing an alignment ofthe INVADER (SEQ ID NO: 50) and probe (SEQ ID NO: 51) along the targetM13mp18 DNA; only 53 bases of the M13mp18 sequence is shown (SEQ ID NO:52). The sequence of the INVADER oligonucleotide is displayed under theM13mp18 target and an arrow is used above the M13mp18 sequence toindicate the position of the INVADER relative to the probe and target.As shown in FIG. 49A, the INVADER and probe oligonucleotides share a 2base region of overlap.

[0894] In FIG. 49B, lanes 1-6 contain reactions performed at 50° C., 55°C., 60° C., and 65° C., respectively; lane 5 contained the controlreaction (lacking target). In FIG. 49B, the products of cleavage areseen as dark bands in the upper half of the panel; the faint lower bandseen appears in proportion to the amount of primary product producedand, while not limiting the invention to a particular mechanism, mayrepresent cleavage one nucleotide into the duplex. The uncleaved probedoes not enter the gel and is thus not visible. The control lane showedno detectable signal over background (lane 5). As expected in aninvasive cleavage reaction, the rate of accumulation of specificcleavage product was temperature-dependent. Using these particularoligonucleotides and target, the fastest rate of accumulation of productwas observed at 55° C. (lane 2) and very little product observed at 65°C. (lane 4).

[0895] When incubated for extended periods at high temperature, DNAprobes can break non-specifically (i.e., suffer thermal degradation) andthe resulting fragments contribute an interfering background to theanalysis. The products of such thermal breakdown are distributed fromsingle-nucleotides up to the full length probe. In this experiment, theability of charge based separation of cleavage products (i.e., chargereversal) would allow the sensitive separation of the specific productsof target-dependent cleavage from probe fragments generated by thermaldegradation was examined.

[0896] To test the sensitivity limit of this detection method, thetarget M13mp18 DNA was serially diluted ten fold over than range of 1fmole to 1 amole. The INVADER and probe oligonucleotides were thosedescribed above (i.e., SEQ ID NOS: 50 and 51). The invasive cleavagereactions were run as described above with the following modifications:the reactions were performed at 55° C., 250 mM or 100 mM KGlu was usedin place of the 100 mM KCl and only 1 pmole of the INVADERoligonucleotide was added. The reactions were initiated as describedabove and allowed to progress for 12.5 hours. A negative controlreaction that lacked added M13m18 target DNA was also run. The reactionswere terminated by the addition of 10 μl of 95% formamide containing 20mM EDTA and 0.02% methyl violet, and 5 μl of these mixtures wereelectrophoresed and visualized as described above. The resulting imagerscan is shown in FIG. 50.

[0897] In FIG. 50, lane 1 contains the negative control; lanes 2-5contain reactions performed using 100 mM KGlu; lanes 6-9 containreactions performed using 250 mM KGlu. The reactions resolved in lanes 2and 6 contained 1 fmole of target DNA; those in lanes 3 and 7 contained100 amole of target; those in lanes 4 and 8 contained 10 amole of targetand those in lanes 5 and 9 contained 1 amole of target. The resultsshown in FIG. 50 demonstrate that the detection limit using chargereversal to detect the production of specific cleavage products in aninvasive cleavage reaction is at or below 1 attomole or approximately6.02×10⁵ target molecules. No detectable signal was observed in thecontrol lane, which indicates that non-specific hydrolysis or otherbreakdown products do not migrate in the same direction asenzyme-specific cleavage products. The excitation and emission maximafor Cy3 are 554 and 568, respectively, while the FMBIO Imager Analyzerexcites at 532 and detects at 585. Therefore, the limit of detection ofspecific cleavage products can be improved by the use of more closelymatched excitation source and detection filters.

Example 24

[0898] Devices and Methods for the Separation and Detection of ChargedReaction Products

[0899] This Example is directed at methods and devices for isolating andconcentrating specific reaction products produced by enzymatic reactionsconducted in solution whereby the reactions generate charged productsfrom either a charge neutral substrate or a substrate bearing theopposite charge borne by the specific reaction product. The methods anddevices of this Example allow isolation of, for example, the productsgenerated by the INVADER-directed cleavage assay of the presentinvention.

[0900] The methods and devices of this Example are based on theprinciple that when an electric field is applied to a solution ofcharged molecules, the migration of the molecules toward the electrodeof the opposite charge occurs very rapidly. If a matrix or otherinhibitory material is introduced between the charged molecules and theelectrode of opposite charge such that this rapid migration isdramatically slowed, the first molecules to reach the matrix will benearly stopped, thus allowing the lagging molecules to catch up. In thisway a dispersed population of charged molecules in solution can beeffectively concentrated into a smaller volume. By tagging the moleculeswith a detectable moiety (e.g., a fluorescent dye), detection isfacilitated by both the concentration and the localization of theanalytes. This Example illustrates two embodiments of devicescontemplated by the present invention; of course, variations of thesedevices will be apparent to those skilled in the art and are within thespirit and scope of the present invention.

[0901]FIG. 51 depicts one embodiment of a device for concentrating thepositively-charged products generated using the methods of the presentinvention. As shown in FIG. 51, the device comprises a reaction tube(10) that contains the reaction solution (11). One end of each of twothin capillaries (or other tubes with a hollow core) (13A and 13B) aresubmerged in the reaction solution (11). The capillaries (13A and 13B)may be suspended in the reaction solution (11) such that they are not incontact with the reaction tube itself; one appropriate method ofsuspending the capillaries is to hold them in place with clamps (notshown). Alternatively, the capillaries may be suspended in the reactionsolution (11) such that they are in contact with the reaction tubeitself. Suitable capillaries include glass capillary tubes commonlyavailable from scientific supply companies (e.g., Fisher Scientific orVWR Scientific) or from medical supply houses that carry materials forblood drawing and analysis. Though the present invention is not limitedto capillaries of any particular inner diameter, tubes with innerdiameters of up to about ⅛ inch (approximately 3 mm) are particularlypreferred for use with the present invention; for example, Kimble No.73811-99 tubes (VWR Scientific) have an inner diameter of 1.1 mm and area suitable type of capillary tube. Although the capillaries of thedevice are commonly composed of glass, any nonconductive tubularmaterial, either rigid or flexible, that can contain either a conductivematerial or a trapping material is suitable for use in the presentinvention. One example of a suitable flexible tube is Tygon® clearplastic tubing (Part No. R3603; inner diameter={fraction (1/16)} inch;outer diameter=⅛ inch).

[0902] As illustrated in FIG. 51, capillary 13A is connected to thepositive electrode of a power supply (20) (e.g., a controllable powersupply available through the laboratory suppliers listed above orthrough electronics supply houses like Radio Shack) and capillary 13B isconnected to the negative electrode of the power supply (20). Capillary13B is filled with a trapping material (14) capable of trapping thepositively-charged reaction products by allowing minimal migration ofproducts that have entered the trapping material (14). Suitable trappingmaterials include, but are not limited to, high percentage (e.g., about20%) acrylamide polymerized in a high salt buffer (0.5 M or highersodium acetate or similar salt); such a high percentage polyacrylamidematrix dramatically slows the migration of the positively-chargedreaction products. Alternatively, the trapping material may comprise asolid, negatively-charged matrix, such as negatively-charged latexbeads, that can bind the incoming positively-charged products. It shouldbe noted that any amount of trapping material (14) capable of inhibitingany concentrating the positively-charged reaction products may be used.Thus, while the capillary 13B in FIG. 51 only contains trapping materialin the lower, submerged portion of the tube, the trapping material (14)can be present in the entire capillary (13B); similarly, less trappingmaterial (14) could be present than that shown in FIG. 51 because thepositively-charged reaction products generally accumulate within a verysmall portion of the bottom of the capillary (13B). The amount oftrapping material need only be sufficient to make contact with thereaction solution (11) and have the capacity to collect the reactionproducts. When capillary 13B is not completely filled with the trappingmaterial, the remaining space is filled with any conductive material(15); suitable conductive materials are discussed below.

[0903] By comparison, the capillary (13A) connected to the positiveelectrode of the power supply 20 may be filled with any conductivematerial (15; indicated by the hatched lines in FIG. 51). This may bethe sample reaction buffer (e.g., 10 mM MOPS, pH 7.5 with 150 mM LiCl, 4mM MnCl₂), a standard electrophoresis buffer (e.g., 45 mM Tris-Borate,pH 8.3, 1.4 mM EDTA), or the reaction solution (11) itself. Theconductive material (15) is frequently a liquid, but a semi-solidmaterial (e.g., a gel) or other suitable material might be easier to useand is within the scope of the present invention. Moreover, thattrapping material used in the other capillary (i.e., capillary 13B) mayalso be used as the conductive material. Conversely, it should be notedthat the same conductive material used in the capillary (13A) attachedto the positive electrode may also be used in capillary 13B to fill thespace above the region containing the trapping material (14) (see FIG.51).

[0904] The top end of each of the capillaries (13A and 13B) is connectedto the appropriate electrode of the power supply (20) by electrode wire(18) or other suitable material. Fine platinum wire (e.g., 0.1 to 0.4mm, Aesar Johnson Matthey, Ward Hill, Mass.) is commonly used asconductive wire because it does not corrode under electrophoresisconditions. The electrode wire (18) can be attached to the capillaries(13A and 13B) by a nonconductive adhesive (not shown), such as thesilicone adhesives that are commonly sold in hardware stores for sealingplumbing fixtures. If the capillaries are constructed of a flexiblematerial, the electrode wire (18) can be secured with a small hose clampor constricting wire (not shown) to compress the opening of thecapillaries around the electrode wire. If the conducting material (15)is a gel, an electrode wire (18) can be embedded directly in the gelwithin the capillary.

[0905] The cleavage reaction is assembled in the reaction tube (10) andallowed to proceed therein as described in proceeding Examples (e.g.,Examples 22-23). Though not limited to any particular volume of reactionsolution (11), a preferred volume is less than 10 ml and more preferablyless than 0.1 ml. The volume need only be sufficient to permit contactwith both capillaries. After the cleavage reaction is completed, anelectric field is applied to the capillaries by turning on the powersource (20). As a result, the positively-charged products generated inthe course of the INVADER-directed cleavage reaction that employs anoligonucleotide, which when cleaved, generates a positively chargedfragment (described in Ex. 23) but when uncleaved bears a net negativecharge, migrate to the negative capillary, where their migration isslowed or stopped by the trapping material (14), and thenegatively-charged uncut and thermally degraded probe molecules migratetoward the positive electrode. Through the use of this or a similardevice, the positively-charged products of the invasive cleavagereaction are separated from the other material (i.e., uncut andthermally degraded probe) and concentrated from a large volume.Concentration of the product in a small amount of trapping material (14)allows for simplicity of detection, with a much higher signal-to-noiseratio than possible with detection in the original reaction volume.Because the concentrated product is labeled with a detectable moietylike a fluorescent dye, a commercially-available fluorescent platereader (not shown) can be used to ascertain the amount of product.Suitable plate readers include both top and bottom laser readers.Capillary 13B can be positioned with the reaction tube (10) at anydesired position so as to accommodate use with either a top or a bottomplate reading device.

[0906] In the alternative embodiment of the present invention depictedin FIG. 52, the procedure described above is accomplished by utilizingonly a single capillary (13B). The capillary (13B) contains the trappingmaterial (14) described above and is connected to an electrode wire(18), which in turn is attached to the negative electrode of a powersupply (20). The reaction tube (10) has an electrode (25) embedded intoits surface such that one surface of the electrode is exposed to theinterior of the reaction tube (10) and another surface is exposed to theexterior of the reaction tube. The surface of the electrode (25) on theexterior of the reaction tube is in contact with a conductive surface(26) connected to the positive electrode of the power supply (20)through an electrode wire (18). Variations of the arrangement depictedin FIG. 52 are also contemplated by the present invention. For example,the electrode (25) may be in contact with the reaction solution (11)through the use of a small hole in the reaction tube (10); furthermore,the electrode wire (18) can be directly attached to the electrode wire(18), thereby eliminating the conductive surface (26).

[0907] As indicated in FIG. 52, the electrode (25) is embedded in thebottom of a reaction tube (10) such that one or more reaction tubes maybe set on the conductive surface (26). This conductive surface couldserve as a negative electrode for multiple reaction tubes; such asurface with appropriate contacts could be applied through the use ofmetal foils (e.g., copper or platinum, Aesar Johnson Matthey, Ward Hill,Mass.) in much the same way contacts are applied to circuit boards.Because such a surface contact would not be exposed to the reactionsample directly, less expensive metals, such as the copper could be usedto make the electrical connections.

[0908] The above devices and methods are not limited to separation andconcentration of positively charged oligonucleotides. As will beapparent to those skilled in the art, negatively charged reactionproducts may be separated from neutral or positively charged reactantsusing the above device and methods with the exception that capillary 13Bis attached to the positive electrode of the power supply (20) andcapillary 13A or alternatively, electrode 25, is attached to thenegative electrode of the power supply (20).

Example 25

[0909] Primer-Directed and Primer Independent Cleavage Occur at the SameSite when the Primer Extends to the 3′ Side of a Mismatched “Bubble” inthe Downstream Duplex

[0910] As discussed above in Example 1, the presence of a primerupstream of a bifurcated duplex can influence the site of cleavage, andthe existence of a gap between the 3′ end of the primer and the base ofthe duplex can cause a shift of the cleavage site up the unpaired 5′ armof the structure (see also Lyamichev et al., supra and U.S. Pat. No.5,422,253). The resulting non-invasive shift of the cleavage site inresponse to a primer is demonstrated in FIGS. 8, 9 and 10, in which theprimer used left a 4-nucleotide gap (relative to the base of theduplex). In FIGS. 8-10, all of the “primer-directed” cleavage reactionsyielded a 21 nucleotide product, while the primer-independent cleavagereactions yielded a 25 nucleotide product. The site of cleavage obtainedwhen the primer was extended to the base of the duplex, leaving no gapwas examined. The results are shown in FIG. 53 (FIG. 53 is areproduction of FIG. 2C in Lyamichev et al. These data were derived fromthe cleavage of the structure shown in FIG. 5, as described inExample 1. Unless otherwise specified, the cleavage reactions comprised0.01 pmoles of heat-denatured, end-labeled hairpin DNA (with theunlabeled complementary strand also present), 1 pmole primer(complementary to the 3′ arm shown in FIG. 5 and having the sequence:5′-GAATTCGATTTAGGTGACAC TATAGAATACA [SEQ ID NO: 53]) and 0.5 units ofDNAPTaq (estimated to be 0.026 pmoles) in a total volume of 10 μl of 10mM Tris-Cl, pH 8.5, and 1.5 mM MgCl₂ and 50 mM KCl. The primer wasomitted from the reaction shown in the first lane of FIG. 53 andincluded in lane 2. These reactions were incubated at 55° C. for 10minutes. Reactions were initiated at the final reaction temperature bythe addition of either the MgCl₂ or enzyme. Reactions were stopped attheir incubation temperatures by the addition of 8 μl of 95% formamidewith 20 mM EDTA and 0.05% marker dyes.

[0911]FIG. 53 is an autoradiogram that indicates the effects on the siteof cleavage of a bifurcated duplex structure in the presence of a primerthat extends to the base of the hairpin duplex. The size of the releasedcleavage product is shown to the left (i.e., 25 nucleotides). Adideoxynucleotide sequencing ladder of the cleavage substrate is shownon the right as a marker (lanes 3-6).

[0912] These data show that the presence of a primer that is adjacent toa downstream duplex (lane 2) produces cleavage at the same site as seenin reactions performed in the absence of the primer (lane 1). (See FIGS.8A and B, 9B and 10A for additional comparisons). When the 3′ terminalnucleotides of the upstream oligonucleotide can base pair to thetemplate strand but are not homologous to the displaced strand in theregion immediately upstream of the cleavage site (i.e., when theupstream oligonucleotide is opening up a “bubble” in the duplex), thesite to which cleavage is apparently shifted is not wholly dependent onthe presence of an upstream oligonucleotide.

[0913] As discussed above in the Background, and in Table 1, therequirement that two independent sequences be recognized in an assayprovides a highly desirable level of specificity. In the invasivecleavage reactions of the present invention, the INVADER and probeoligonucleotides must hybridize to the target nucleic acid with thecorrect orientation and spacing to enable the production of the correctcleavage product. When the distinctive pattern of cleavage is notdependent on the successful alignment of both oligonucleotides in thedetection system these advantages of independent recognition are lost.

Example 26

[0914] Invasive Cleavage and Primer-Directed Cleavage when there is onlyPartial Homology in the “X” Overlap Region

[0915] While not limiting the present invention to any particularmechanism, invasive cleavage occurs when the site of cleavage is shiftedto a site within the duplex formed between the probe and the targetnucleic acid in a manner that is dependent on the presence of anupstream oligonucleotide that shares a region of overlap with thedownstream probe oligonucleotide. In some instances, the 5′ region ofthe downstream oligonucleotide may not be completely complementary tothe target nucleic acid. In these instances, cleavage of the probe mayoccur at an internal site within the probe even in the absence of anupstream oligonucleotide (in contrast to the base-by-base nibbling seenwhen a fully paired probe is used without an INVADER). Invasive cleavageis characterized by an apparent shifting of cleavage to a site within adownstream duplex that is dependent on the presence of the INVADERoligonucleotide.

[0916] A comparison between invasive cleavage and primer-directedcleavage may be illustrated by comparing the expected cleavage sites ofa set of probe oligonucleotides having decreasing degrees ofcomplementarity to the target strand in the 5′ region of the probe(i.e., the region that overlaps with the INVADER). A simple test,similar to that performed on the hairpin substrate above (Ex. 25), canbe performed to compare invasive cleavage with the non-invasiveprimer-directed cleavage described above. Such a set of testoligonucleotides is diagrammed in FIG. 54. The structures shown in FIG.54 are grouped in pairs, labeled “a”, “b”, “c”, and “d”. Each pair hasthe same probe sequence annealed to the target strand (SEQ ID NO: 54),but the top structure of each pair is drawn without an upstreamoligonucleotide, while the bottom structure includes thisoligonucleotide (SEQ ID NO: 55). The sequences of the probes shown inFIGS. 54a-54 d are listed in SEQ ID NOS:32, 56, 57 and 58, respectively.Probable sites of cleavage are indicated by the black arrowheads. (It isnoted that the precise site of cleavage on each of these structures mayvary depending on the choice of cleavage agent and other experimentalvariables. These particular sites are provided for illustrative purposesonly.) To conduct this test, the site of cleavage of each probe isdetermined both in the presence and the absence of the upstreamoligonucleotide, in reaction conditions such as those described inExample 18. The products of each pair of reactions are then be comparedto determine whether the fragment released from the 5′ end of the probeincreases in size when the upstream oligonucleotide is included in thereaction.

[0917] The arrangement shown in FIG. 54a, in which the probe molecule iscompletely complementary to the target strand, is similar to that shownin FIG. 28. Treatment of the top structure with the 5′ nuclease of a DNApolymerase would cause exonucleolytic nibbling of the probe (i.e., inthe absence of the upstream oligonucleotide). In contrast, inclusion ofan INVADER oligonucleotide would cause a distinctive cleavage shiftsimilar, to those observed in FIG. 29.

[0918] The arrangements shown in FIGS. 54b and 54 c have some amount ofunpaired sequence at the 5′ terminus of the probe ( 3 and 5 bases,respectively). These small 5′ arms are suitable cleavage substrate forthe 5′ nucleases and would be cleaved within 2 nucleotide's of thejunction between the single stranded region and the duplex. In thesearrangements, the 3′ end of the upstream oligonucleotide shares identitywith a portion of the 5′ region of the probe that is complementary tothe target sequence (that is the 3′ end of the INVADER has to competefor binding to the target with a portion of the 5′ end of the probe).Therefore, when the upstream oligonucleotide is included it is thoughtto mediate a shift in the site of cleavage into the downstream duplex(although the present invention is not limited to any particularmechanism of action), and this would, therefore, constitute invasivecleavage. If the extreme 5′ nucleotides of the unpaired region of theprobe were able to hybridize to the target strand, the cleavage site inthe absence of the INVADER might change but the addition of the INVADERoligonucleotide would still shift the cleavage site to the properposition.

[0919] Finally, in the arrangement shown in FIG. 54d, the probe andupstream oligonucleotides share no significant regions of homology, andthe presence of the upstream oligonucleotide would not compete forbinding to the target with the probe. Cleavage of the structures shownin FIG. 54d would occur at the same site with or without the upstreamoligonucleotide, and is thus would not constitute invasive cleavage.

[0920] By examining any upstream oligonucleotide/probe pair in this way,it can easily be determined whether the resulting cleavage is invasiveor merely primer-directed. Such analysis is particularly useful when theprobe is not fully complementary to the target nucleic acid, so that theexpected result may not be obvious by simple inspection of thesequences.

Example 27

[0921] Modified CLEAVASE Enzymes

[0922] In order to develop nucleases having useful activities for thecleavage of nucleic acids the following modified nucleases wereproduced.

[0923] a) CLEAVASE BN/thrombin Nuclease

[0924] i) Cloning and Expression of CLEAVASE BN/Thrombin Nuclease

[0925] Site directed mutagenesis was used to introduce a proteinsequence recognized by the protease thrombin into the region of theCLEAVASE BN nuclease that is thought to form the helical arch of theprotein through which the single-stranded DNA that is cleaved mustpresumably pass. Mutagenesis was carried out using the Transformer™mutagenesis kit (Clonetech, Palo Alto, Calif.) according tomanufacturer's protocol using the mutagenic oligonucleotide5′-GGGAAAGTCCTCGCAGCCGCGCG GGACGAGCGTGGGGGCCCG (SEQ ID NO: 59). Aftermutagenesis, the DNA was sequenced to verify the insertion of thethrombin cleavage site. The DNA sequence encoding the CLEAVASEBN/thrombin nuclease is provided in SEQ ID NO: 60; the amino acidsequence of CLEAVASE BN/thrombin nuclease is provided in SEQ ID NO: 61.

[0926] A large scale preparation of the thrombin mutant (i.e., CLEAVASEBN/thrombin) was done using E. coli cells overexpressing the CLEAVASEBN/thrombin nuclease as described in Example 28.

[0927] ii) Thrombin Cleavage of CLEAVASE BN/Thrombin

[0928] Six point four (6.4) mg of the purified CLEAVASE BN/thrombinnuclease was digested with 0.4 U of thrombin (Novagen) for 4 hours at23° C. or 37° C. Complete digestion was verified by electrophoresis on a15% SDS polyacrylamide gel followed by staining with Coomassie BrilliantBlue R. Wild-type CLEAVASE BN nuclease was also digested with thrombinas a control. The resulting gel is shown in FIG. 61.

[0929] In FIG. 61, lane 1 contains molecular weight markers (Low-RangeProtein Molecular Weight Markers; Promega), lane 2 contains undigestedCLEAVASE BN/throbin nuclease, lanes 3 and 4 contain CLEAVASE BN/thrombinnuclease digested with thrombin at 23° C. for 2 and 4 hours,respectively, and lanes 5 and 6 contain CLEAVASE BN/thrombin nucleasedigested with thrombin at 37° C. for 2 and 4 hours, respectively. Theseresults show that the CLEAVASE BN/thrombin nuclease has an apparentmolecular weight of 36.5 kilodaltons and demonstrate that CLEAVASEBN/thrombin nuclease is efficiently cleaved by thrombin. In addition,the thrombin cleavage products have approximate molecular weights of 27kilodaltons and 9 kilodaltons, the size expected based upon the positionof the inserted thrombin site in the CLEAVASE BN/thrombin nuclease.

[0930] To determine the level of hairpin cleavage activity in digestedand undigested CLEAVASE BN/thrombin nuclease, dilutions were made andused to cleave a test hairpin containing a 5′ fluoroscein label. Varyingamounts of digested and undigested CLEAVASE BN/thrombin nuclease wereincubated with 5 μM oligonucleotide S-60 hairpin (SEQ ID NO: 29; seeFIG. 26) in 10 mM MOPS (pH 7.5), 0.05% Tween-20, 0.05% NP-40, and 1 mMMnCl₂ for 5 minutes at 60° C. The digested mixture was electrophoresedon a 20% acrylamide gel and visualized on a Hitachi FMBIO 100fluoroimager. The resulting image is shown in FIG. 62.

[0931] In FIG. 62, lane 1 contains the no enzyme control, lane 2contains reaction products produced using 0.01 ng of CLEAVASE BNnuclease, lanes 3, 4, and 5 contain reaction products produced using0.01 ng, 0.04 ng, and 4 ng of undigested CLEAVASE BN/thrombin nuclease,respectively, and lanes 6, 7, and 8 contain reaction products producedusing 0.01 ng, 0.04 ng, and 4 ng of thrombin-digested CLEAVASEBN/thrombin nuclease, respectively. The results shown in FIG. 62demonstrated that the insertion of the thrombin cleavage site reducedcleavage activity about 200-fold (relative to the activity of CLEAVASEBN nuclease), but that digestion with thrombin did not reduce theactivity significantly.

[0932] M13 single-stranded DNA was used as a substrate for cleavage byCLEAVASE BN nuclease and digested and undigested CLEAVASE BN/thrombinnuclease. Seventy nanograms of single-stranded M13 DNA (NEB) wasincubated in 10 mM MOPS, pH 7.5, 0.05% Tween-20, 0.05% NP-40, 1 mM MgCl₂or 1 mM MnCl₂, with 8 ng of CLEAVASE BN nuclease, undigested CLEAVASEBN/thrombin nuclease, or digested CLEAVASE BN/thrombin nuclease for 10minutes at 50° C. Reaction mixtures were electrophoresed on a 0.8%agarose gel and then stained with a solution containing 0.5 μg/mlethidium bromide (EtBr) to visualize DNA bands. A negative image of theEtBr-stained gel is shown in FIG. 63.

[0933] In FIG. 63, lane 1 contains the no enzyme control, lane 2contains reaction products produced using CLEAVASE BN nuclease and 1 mMMgCl₂, lane 3 contains reaction products produced using CLEAVASE BNnuclease and 1 mM MnCl₂, lane 4 contains reaction products producedusing undigested CLEAVASE BN/thrombin nuclease and 1 mM MgCl₂, lane 5contains reaction products produced using undigested CLEAVASEBN/thrombin nuclease and 1 mM MnCl₂, lane 6 contains reaction productsproduced using thrombin-digested CLEAVASE BN/thrombin nuclease and 1 mMMgCl₂, and lane 7 contains reaction products produced usingthrombin-digested CLEAVASE BN/thrombin nuclease and 1 mM MnCl₂. Theresults shown in FIG. 63 demonstrated that the CLEAVASE BN/thrombinnuclease had an enhanced ability to cleave circular DNA (and thus areduced requirement for the presence of a free 5′ end) as compared tothe CLEAVASE BN nuclease.

[0934] It can be seen from these data that the helical arch of theseproteins can be opened without destroying the enzyme or its ability tospecifically recognize cleavage structures. The CLEAVASE BN/thrombinmutant has an increased ability to cleave without reference to a 5′ end,as discussed above. The ability to cleave such structures will allow thecleavage of long molecules, such as genomic DNA that, while often notcircular, may present many desirable cleavage sites that are at a farremoved from any available 5′ end. Cleavage structures may be made atsuch sites either by folding of the strands (i.e., CFLP® cleavage) or bythe introduction of structure-forming oligonucleotides (U.S. Pat. No.5,422,253). 5′ ends of nucleic acids can also be made unavailablebecause of binding of a substance too large to thread through thehelical arch. Such binding moieties may include proteins such asstreptavidin or antibodies, or solid supports such as beads or the wallsof a reaction vessel. A cleavage enzyme with an opening in the loop ofthe helical arch will be able to cleave DNAs that are configured in thisway, extending the number of ways in which reactions using such enzymescan be formatted.

[0935] b) CLEAVASE DN Nuclease

[0936] i) Construction and Expression of CLEAVASE DN Nuclease

[0937] A polymerization deficient mutant of Taq DNA polymerase, termedCLEAVASE DN nuclease, was constructed. CLEAVASE DN nuclease contains anasparagine residue in place of the wild-type aspartic acid residue atposition 785 (D785N).

[0938] DNA encoding the CLEAVASE DN nuclease was constructed from thegene encoding for CLEAVASE A/G (mutTaq, Ex. 2) in two rounds ofsite-directed mutagenesis. First, the G at position 1397 and the G atposition 2264 of the CLEAVASE A/G gene (SEQ ID NO: 21) were changed to Aat each position to recreate a wild-type DNAPTaq gene. As a second roundof mutagenesis, the wild type DNAPTaq gene was converted to the CLEAVASEDN gene by changing the G at position 2356 to A. These manipulationswere performed as follows.

[0939] DNA encoding the CLEAVASE A/G nuclease was recloned from pTTQ18plasmid (Ex. 2) into the pTrc99A plasmid (Pharmacia) in a two stepprocedure. First, the pTrc99A vector was modified by removing the G atposition 270 of the pTrc99A map, creating the pTrc99G cloning vector. Tothis end, pTrc99A plasmid DNA was cut with NcoI and the recessive 3′ends were filled-in using the Klenow fragment of E. coli polymerase I inthe presence of all four dNTPs at 37° C. for 15 min. After inactivationof the Klenow fragment by incubation at 65° C. for 10 min, the plasmidDNA was cut with EcoRI, the ends were again filled-in using the Klenowfragment in the presence of all four dNTPs at 37° C. for 15 min. TheKlenow fragment was then inactivated by incubation at 65° C. for 10 min.The plasmid DNA was ethanol precipitated, recircularized by ligation,and used to transform E. coli JM109 cells (Promega). Plasmid DNA wasisolated from single colonies and deletion of the G at position 270 ofthe pTrc99A map was confirmed by DNA sequencing.

[0940] As a second step, DNA encoding the CLEAVASE A/G nuclease wasremoved from the pTTQ18 plasmid using EcoRI and SalI and the DNAfragment carrying the CLEAVASE A/G nuclease gene was separated on a 1%agarose gel and isolated with Geneclean II Kit (Bio 101, Vista, Calif.).The purified fragment was ligated into the pTrc99G vector, which hadbeen cut with EcoRI and SalI. The ligation mixture was used to transformcompetent E. coli JM109 cells (Promega). Plasmid DNA was isolated fromsingle colonies and insertion of the CLEAVASE A/G nuclease gene wasconfirmed by restriction analysis using EcoRI and SalI.

[0941] Plasmid DNA pTrcAG carrying the CLEAVASE A/G nuclease gene clonedinto the pTrc99A vector was purified from 200 ml of JM109 overnightculture using QIAGEN Plasmid Maxi kit (QIAGEN, Chatsworth, Calif.)according to manufacturer's protocol. pTrcAG plasmid DNA was mutagenizedusing two mutagenic primers, E465 (SEQ ID NO: 62) (Integrated DNATechnologies, Iowa) and R754Q (SEQ ID NO: 63) (Integrated DNATechnologies), and the selection primer Trans Oligo AlwNI/SpeI(Clontech, Palo Alto, Calif., catalog #6488-1) according to Transformer™Site-Directed Mutagenesis Kit protocol (Clontech) to produce a restoredwild-type DNAPTaq gene (pTrcWT).

[0942] pTrcWT plasmid DNA carrying the wild-type DNAPTaq gene clonedinto the pTrc99A vector was purified from 200 ml of JM109 overnightculture using QIAGEN Plasmid Maxi kit (QIAGEN, Chatsworth, Calif.)according to manufacturer's protocol. pTrcWT was then mutagenized usingthe mutagenic primer D785N (SEQ ID NO: 64) (Integrated DNA Technologies)and the selection primer Switch Oligo SpeI/AlwNI (Clontech, catalog#6373-1) according to Transformer™ Site-Directed Mutagenesis Kitprotocol (Clontech) to create a plasmid containing DNA encoding theCLEAVASE DN nuclease. The DNA sequence encoding the CLEAVASE DN nucleaseis provided in SEQ ID NO: 65; the amino acid sequence of CLEAVASE DNnuclease is provided in SEQ ID NO: 66.

[0943] A large scale preparation of the CLEAVASE DN nuclease was doneusing E. coli cells overexpressing the CLEAVASE DN nuclease as describedin Example 28.

[0944] c) CLEAVASE DA Nuclease and CLEAVASE DV Nuclease

[0945] Two polymerization deficient mutants of Taq DNA polymerase,termed CLEAVASE DA nuclease and CLEAVASE DV nuclease, were constructed.The CLEAVASE DA nuclease contains a alanine residue in place of thewild-type aspartic acid residue at position 610 (D785A). The CLEAVASE DVnuclease contains a valine residue in place of the wild-type asparticacid residue at position 610 (D610V).

[0946] i) Construction and Expression of the CLEAVASE DA and CLEAVASE DVNucleases

[0947] To construct vectors encoding the CLEAVASE DA and DV nucleases,the CLEAVASE A/G nuclease gene contained within pTrcAG was mutagenizedwith two mutagenic primers, R754Q (SEQ ID NO: 63) and D610AV (SEQ ID NO:67) and the selection primer Trans Oligo AlwNI/SpeI (Clontech, catalog#6488-1) according to the Transformer™ Site-Directed Mutagenesis Kitprotocol (Clontech,) to create a plasmid containing DNA encoding theCLEAVASE DA nuclease or CLEAVASE DV nuclease. The D610AV oligonucleotidewas synthesized to have a purine, A or G, at position 10 from the 5′ endof the oligonucleotide. Following mutagenesis, plasmid DNA was isolatedfrom single colonies and the type of mutation present, DA or DV, wasdetermined by DNA sequencing. The DNA sequence encoding the CLEAVASE DAnuclease is provided in SEQ ID NO: 68; the amino acid sequence ofCLEAVASE DA nuclease is provided in SEQ ID NO: 69. The DNA sequenceencoding the CLEAVASE DV nuclease is provided in SEQ ID NO: 70; theamino acid sequence of CLEAVASE DV nuclease is provided in SEQ ID NO:71.

[0948] Large scale preparations of the CLEAVASE DA and CLEAVASE DVnucleases was done using E. coli cells overexpressing the CLEAVASE DAnuclease or the CLEAVASE DV nuclease as described in Example 28.

Example 28

[0949] Cloning and Expression of Thermostable FEN-1 Endonucleases

[0950] Sequences encoding thermostable FEN-1 proteins derived from threeArchaebacterial species were cloned and overexpressed in E. coli. ThisExample involved a) cloning and expression of a FEN-1 endonuclease fromMethanococcus jannaschii; b) cloning and expression of a FEN-1endonuclease from Pyrococcus furiosus; c) cloning and expression of aFEN-1 endonuclease from Pyrococcus woesei; d) cloning and expression ofa FEN-1 endonuclease from Archaeoglobus fulgidus; e) large scalepreparation of recombinant thermostable FEN-1 proteins; and f) activityassays using FEN-1 endonucleases.

[0951] a) Cloning and Expression of a FEN-1 Endonuclease fromMethanococcus jannaschii

[0952] DNA encoding the FEN-1 endonuclease from Methanococcus jannaschii(M. jannaschii) was isolated from M. jannaschii cells and inserted intoa plasmid under the transcriptional control of an inducible promoter asfollows. Genomic DNA was prepared from 1 vial of live M. jannaschiibacteria (DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen,Braunschweig, Germany #2661) with the DNA XTRAX kit (Gull Laboratories,Salt Lake City, Utah) according to the manufacturer's protocol. Thefinal DNA pellet was resuspended in 100 μl of TE (10 mM Tris HCl, pH8.0, 1 mM EDTA). One microliter of the DNA solution was employed in aPCR using the Advantage™ cDNA PCR kit (Clonetech); the PCR was conductedaccording to manufacturer's recommendations. The 5′-end primer (SEQ IDNO: 72) is complementary to the 5′ end of the Mja FEN-1 open readingframe with a one base substitution to create an NcoI restriction site (afragment of the M. jannaschii genome that contains the gene encoding M.jannaschii (Mja) FEN-1 is available from GenBank as accession #U67585).The 3′-end primer (SEQ ID NO: 73) is complementary to a sequence about15 base pairs downstream from the 3′ end of the Mja FEN-1 open readingframe with 2 base substitutions to create a SalI restriction enzymesite. The sequences of the 5′-end and 3′-end primers are: 5′-GGGATACCATGGGAGTGCAGTTTGG-3′ (SEQ ID NO: 72) and 5′-GGTAAATTTTTCTCGTCGACATCCCAC-3′ (SEQ ID NO: 73), respectively. The PCR reaction resulted inthe amplification (i.e., production) of a single major band about 1kilobase in length. The open reading frame (ORF) encoding the Mja FEN-1endonuclease is provided in SEQ ID NO: 74; the amino acid sequenceencoded by this ORF is provided in SEQ ID NO: 75.

[0953] Following the PCR amplification, the entire reaction waselectrophoresed on a 1.0% agarose gel and the major band was excisedfrom the gel and purified using the Geneclean II kit (Bio101, Vista,Calif.) according to manufacturer's instructions. Approximately 1 μg ofthe gel-purified Mja FEN-1 PCR product was digested with NcoI and SalI.After digestion, the DNA was purified using the Geneclean II kitaccording to manufacturer's instructions. One microgram of the pTrc99avector (Pharmacia) was digested with NcoI and SalI in preparation forligation with the digested PCR product. One hundred nanograms ofdigested pTrc99a vector and 250 ng of digested Mja FEN-1 PCR productwere combined and ligated to create pTrc99-MJFEN1. pTrc99-MJFEN1 wasused to transform competent E. coli JM109 cells (Promega) using standardtechniques.

[0954] b) Cloning and Expression of a FEN-1 Endonuclease from Pyrococcusfuriosus

[0955] DNA encoding the Pyrococcus furiosus (P. furiosus) FEN-1endonuclease was obtained by PCR amplification using a plasmidcontaining DNA encoding the P. furiosus (Pfu) FEN-1 endonuclease(obtained from Dr. Frank Robb, Center of Marine Biotechnology,Baltimore, Md.). DNA sequences encoding a portion of the Pfu FEN-1endonuclease can be obtained from GenBank as accession Nos. AA113505 andW36094. The amplified Pfu FEN-1 gene was inserted into the pTrc99aexpression vector (Pharmacia) to place the Pfu FEN-1 gene under thetranscriptional control of the inducible trc promoter. The PCRamplification was conducted as follows. One hundred microliter reactionscontained 50 mM Tris HCl, pH 9.0, 20 mM (NH₄)₂SO₄, 2 mM MgCl₂, 50 μMdNTPs, 50 pmole each primer, 1 U Tfl polymerase (Epicentre Technologies,Madison, Wis.) and 1 ng of FEN-1 gene-containing plasmid DNA. The 5′-endprimer (SEQ ID NO: 76) is complementary to the 5′ end of the Pfu FEN-1open reading frame but with two substitutions to create an NcoI site andthe 3′ -end primer (SEQ ID NO: 77) is complementary to a region locatedabout 30 base pairs downstream of the FEN-1 open reading frame with twosubstitutions to create a PstI site. The sequences of the 5′-end and3′-end primers are: 5′-GAGGTGATACCATG GGTGTCC-3′ (SEQ ID NO: 76) and5′-GAAACTCTGCAGCGCGTCAG-3′ (SEQ ID NO: 77), respectively. The PCRreaction resulted in the amplification of a single major band about 1kilobase in length. The open reading frame (ORF) encoding the Pfu FEN-1endonuclease is provided in SEQ ID NO: 78; the amino acid sequenceencoded by this ORF is provided in SEQ ID NO: 79.

[0956] Following the PCR amplification, the entire reaction waselectrophoresed on a 1.0% agarose gel and the major band was excisedfrom the gel and purified using the Geneclean II kit (Bio101) accordingto manufacturer's instructions. Approximately 1 μg of gel purified PfuFEN-1 PCR product was digested with NcoI and PstI. After digestion, theDNA was purified using the Geneclean II kit according to manufacturer'sinstructions. One microgram of the pTrc99a vector was digested with NcoIand PstI prior to ligation with the digested PCR product. One hundrednanograms of digested pTrc99a and 250 ng of digested Pfu FEN-1 PCRproduct were combined and ligated to create pTrc99-PFFEN1. pTrc99-PFFEN1was used to transform competent E. coli JM109 cells (Promega) usingstandard techniques.

[0957] c) Cloning and Expression of a FEN-1 Endonuclease from Pyrococcuswoesei

[0958] For the cloning of DNA encoding the Pyrococcus woesei (Pwo) FEN-1endonuclease, DNA was prepared from lyophilized P. woesei bacteria (DSMZ#3773) as described (Zwickl et al., J. Bact., 172:4329 [1990]) withseveral changes. Briefly, one vial of P. woesei bacteria was rehydratedand resuspended in 0.5 ml of LB (Luria broth). The cells werecentrifuged at 14,000×g for 1 min and the cell pellet was resuspended in0.45 ml of TE. Fifty microliters of 10% SDS was added and the mixturewas incubated at RT for 5 min. The cell lysate was then extracted threetime with 1:1 phenol:chloroform and three times with chloroform. Fivehundred microliters of isopropanol was added to the extracted lysate andthe DNA was pelleted by centrifugation at 14,000×g for 10 min. The DNApellet was washed in 0.5 ml of 70% ethanol and the DNA was pelletedagain by centrifugation at 14,000×g for 5 min. The DNA pellet was driedand resuspended in 100 μl of TE and used for PCR reactions withoutfurther purification.

[0959] To generate a P. woesei FEN-1 gene fragment for cloning into anexpression vector, low stringency PCR was attempted with primerscomplementary to the ends of the P. furiosus FEN-1 gene open readingframe. The sequences of the 5′-end and 3′-end primers are5′-GATACCATGGGTGTCCCAATTGGTG-3′ (SEQ ID NO: 80) and5′-TCGACGTCGACTTATCTCTTGAACCAACTTTCAAGGG-3′ (SEQ ID NO: 81),respectively. The high level of sequence similarity of protein homologs(i.e., proteins other than FEN-1 proteins) from P. furiosus and P.woesei suggested that there was a high probability that the P. woeseiFEN-1 gene could be amplified using primers containing sequencescomplementary to the P. furiosus FEN-1 gene. However, this approach wasunsuccessful under several different PCR conditions.

[0960] The DNA sequence of FEN-1 genes from P. furiosus and M.jannaschii were aligned and blocks of sequence identity between the twogenes were identified. These blocks were used to design internal primers(i.e., complementary to sequences located internal to the 5′ and 3′ endsof the ORF) for the FEN-1 gene that are complementary to the P. furiosusFEN-1 gene in those conserved regions. The sequences of the 5′- and3′-internal primers are 5′-AGCGAGGGAGAGGCCCAAGC-3′ (SEQ ID NO: 82) and5′-GCCTATGCCCTTTATTCCTCC-3′ (SEQ ID NO: 83), respectively. A PCRemploying these internal primers was conducted using the Advantage™ PCRkit and resulted in production of a major band of ˜300 bp.

[0961] Since the PCR with the internal primers was successful, reactionswere attempted that contained mixtures of the internal (SEQ ID NOS:82and 83) and external (SEQ ID NOS:80 and 81) primers. A reactioncontaining the 5′-end external primer (SEQ ID NO: 80) and 3′-endinternal primer (SEQ ID NO: 83) resulted in the production of a 600 bpband and a reaction containing the 5′-end internal primer (SEQ ID NO:82) and 3′-end external primer (SEQ ID NO: 81) resulted in theproduction of a 750 bp band. These overlapping DNA fragments weregel-purified and combined with the external primers (SEQ ID NOS:80 and81) in a PCR reaction. This reaction generated a 1 kb DNA fragmentcontaining the entire Pwo FEN-1 gene open reading frame. The resultingPCR product was gel-purified, digested, and ligated exactly as describedabove for the Mja FEN-1 gene PCR product. The resulting plasmid wastermed pTrc99-PWFEN1. pTrc99-PWFEN1 was used to transform competent E.coli JM109 cells (Promega) using standard techniques.

[0962] d) Cloning and Expression of a FEN-1 Endonuclease fromArchaeoglobus fulgidus

[0963] The preliminary Archaeoglobus fulgidus (Afu) chromosome sequenceof 2.2 million bases was downloaded from the TIGR (The Institute forGenomic Research) world wide web site, and imported into a softwareprogram (MacDNAsis), used to analyze and manipulate DNA and proteinsequences. The unannotated sequence was translated into all 6 of thepossible reading frames, each comprising approximately 726,000 aminoacids. Each frame was searched individually for the presence of theamino acid sequence “VFDG” (valine, phenylalanine, aspartic acid,glycine), a sequence that is conserved in the FEN-1 family. The aminoacid sequence was found in an open reading frame that contained otheramino acid sequences conserved in the FEN-1 genes and that wasapproximately the same size as the other FEN-1 genes. The ORF DNAsequence is shown in SEQ ID NO: 164, while the ORF protein sequence isshown in SEQ ID NO: 165. Based on the position of this amino acidsequence within the reading frame, the DNA sequence encoding a putativeFEN-1 gene was identified.

[0964] The sequence information was used to design oligonucleotideprimers that were used for PCR amplification of the FEN-1-like sequencefrom A. fulgidus genomic DNA. Genomic DNA was prepared from A. fulgidusas described in Ex. 29a for M. janaschii, except that one vial(approximately 5 ml of culture) of live A. fulgidus bacteria from DSMZ(DSMZ #4304) was used. One microliter of the genomic DNA was used forPCR reaction as described in Ex. 29a. The 5′ end primer is complementaryto the 5′ end of the Afu FEN-1 gene except it has a 1 base pairsubstitution to create an Nco I site. The 3′ end primer is complentaryto the 3′ end of the Afu FEN-1 gene downstream from the FEN-1 ORF exceptit contains a 2 base substitution to create a Sal I site. The sequencesof the 5′ and 3′ end primers are 5′-CCGTCAACATTTACCATGGGTGCGGA-3′ (SEQID NO: 166) and 5′-CCGCCACCTCGTAGTCGACATCCTTTTCGTG (SEQ ID NO: 167),respectively.

[0965] Cloning of the resulting fragment was as described for thePfuFEN1 gene, above, to create the plasmid pTrc99-AFFEN1. The pTrcAfuHisplasmid was constructed by modifying pTrc99-AFFEN1, by adding ahistidine tail to facilitate purification. To add this histidine tail,standard PCR primer-directed mutagenesis methods were used to insert thecoding sequence for six histidine residues between the last amino acidcodon of the pTrc99-AFFEN1 coding region and the stop codon. Theresulting plasmid was termed pTrcAfuHis. The protein was then expressedas described in Example 28(e), and purified by binding to a Ni++affinity column, as described in Example 8.

[0966] e) Large Scale Preparation of Recombinant Thermostable FEN-1Proteins

[0967] The Mja, Pwo and Pfu FEN-1 proteins were purified by thefollowing technique, which is derived from a Taq DNA polymerasepreparation protocol (Engelke et al., Anal. Biochem., 191:396 [1990]) asfollows. E. coli cells (strain JM109) containing either pTrc99-PFFEN1,pTrc99-PWFEN1, or pTrc99-MJFEN1 were inoculated into 3 ml of LB (LuriaBroth) containing 100 μg/ml ampicillin and grown for 16 hrs at 37° C.The entire overnight culture was inoculated into 200 ml or 350 ml of LBcontaining 100 μg/ml ampicillin and grown at 37° C. with vigorousshaking to an A₆₀₀ of 0.8. lPTG (1 M stock solution) was added to afinal concentration of 1 mM and growth was continued for 16 hrs at 37°C.

[0968] The induced cells were pelleted and the cell pellet was weighed.An equal volume of 2× DG buffer (100 mM Tris-HCl, pH 7.6, 0.1 mM EDTA)was added and the pellet was resuspended by agitation. Fifty mg/mllysozyme (Sigma, St. Louis, Mo.) was added to 1 mg/ml finalconcentration and the cells were incubated at room temperature for 15min. Deoxycholic acid (10% solution) was added dropwise to a finalconcentration of 0.2 % while vortexing. One volume of H₂O and 1 volumeof 2× DG buffer was added and the resulting mixture was sonicated for 2minutes on ice to reduce the viscosity of the mixture. After sonication,3 M (NH₄)₂SO₄ was added to a final concentration of 0.2 M and the lysatewas centrifuged at 14000×g for 20 min at 4° C. The supernatant wasremoved and incubated at 70° C. for 60 min at which time 10%polyethylimine (PEI) was added to 0.25%. After incubation on ice for 30min., the mixture was centrifuged at 14,000×g for 20 min at 4° C. Atthis point, the supernatant was removed and the FEN-1 proteins wasprecipitated by the addition of (NH₄)₂SO₄ as follows.

[0969] For the Pwo and the Pfu FEN-1 preparations, the FEN-1 protein wasprecipitated by the addition of 2 volumes of 3 M (NH₄)₂SO₄. The mixturewas incubated overnight at room temperature for 16 hrs and the proteinwas centrifuged at 14,000×g for 20 min at 4° C. The protein pellet wasresuspended in 0.5 ml of Q buffer (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA,0.1% Tween 20). For the Mja FEN-1 preparation, solid (NH₄)₂SO₄ was addedto a final concentration of 3 M (˜75% saturated), the mixture wasincubated on ice for 30 min, and the protein was spun down andresuspended as described above.

[0970] The resuspended protein preparations were quantitated bydetermination of the A₂₇₉ and aliquots containing 2-4 μg of totalprotein were electrophoresed on a 10 % SDS polyacrylamide gel (29:1acrylamide: bis-acrylamide) in standard Laemmli buffer [Laemmli, Nature277:680 [1970]) and stained with Coomassie Brilliant Blue R; the resultsare shown in FIG. 64.

[0971] In FIG. 64, lane 1 contains molecular weight markers (Mid-RangeProtein Molecular Weight Markers; Promega); the size of the markerproteins is indicated to the left of the gel. Lane 2 contains purifiedCLEAVASE BN nuclease; lanes 3-5 contain extracts prepared from E. coliexpressing the Pfu, Pwo and Mja FEN-1 nucleases, respectively. Thecalculated (i.e., using a translation of the DNA sequence encoding thenuclease) molecular weight of the Pfu FEN-1 nuclease is 38,714 daltonsand the calculated molecular weight for the Mja FEN-1 nuclease is 37,503Daltons. The Pwo and Pfu FEN-1 proteins co-migrated on the SDS-PAGE geland therefore, the molecular weight of the Pwo FEN-1 nuclease wasestimated to be 38.7 kDa.

[0972] f) Activity Assays Using FEN-1 Endonucleases

[0973] i) Mixed Hairpin Assay

[0974] The CLEAVASE BN nuclease has an approximately 60-fold greateraffinity for a 12 base pair stem-loop structure than an 8 base pairstem-loop DNA structure. As a test for activity differences between theCLEAVASE BN nuclease and the FEN-1 nucleases, a mixture ofoligonucleotides having either a 8 or a 12 bp stem-loop (see FIG. 60,which depicts the S-33 and 11-8-0 oligonucleotides) was incubated withan extract prepared from E. coli cells overexpressing the Mja FEN-1nuclease (prepared as described above). Reactions contained 0.05 μM ofoligonucleotides S-33 (SEQ ID NO: 84) and 11-8-0 (SEQ ID NO: 85) (botholigonucleotides contained 5′-fluorescein labels), 10 mM MOPS, pH 7.5,0.05% Tween-20, 0.05% NP-40, 1 mM MnCl₂. Reactions were heated to 90° C.for 10 seconds, cooled to 55° C., then 1 μl of crude extract (Mja FEN-1)or purified enzyme (CLEAVASE BN nuclease) was added and the mixtureswere incubated at 55° C. for 10 minutes; a no enzyme control was alsorun. The reactions were stopped by the addition of formamide/EDTA, thesamples were electrophoresed on a denaturing 20% acrylamide gel andvisualized on a Hitachi FMBIO 100 fluoroimager. The resulting image isshown in FIG. 65.

[0975] In FIG. 65, lane 1 contains the reaction products generated bythe CLEAVASE BN nuclease, lane 2 contains the reaction products from theno enzyme control reaction and lane 3 contains the reaction productsgenerated by the Mja FEN-1 nuclease. The data shown in FIG. 76demonstrates that the CLEAVASE BN nuclease strongly prefers the S33structure (12 bp stem-loop) while the Mja FEN-1 nuclease cleavesstructures having either an 8 or a 12 bp stem-loop with approximatelythe same efficiency. This shows that the Mja FEN-1 nuclease has adifferent substrate specificity than the CLEAVASE BN nuclease, a usefulfeature for INVADER assays or CFLP® analysis as discussed in theDescription of the Invention.

Example 29

[0976] Terminal Deoxynucleotidyl Transferase Selectively Extends theProducts of INVADER-Directed Cleavage

[0977] The majority of thermal degradation products of DNA probes willhave a phosphate at the 3′-end. To investigate if thetemplate-independent DNA polymerase, terminal deoxynucleotidetransferase (TdT) can tail or polymerize the aforementioned 3′-endphosphates (i.e., add nucleotide triphosphates to the 3′ end) thefollowing experiment was performed.

[0978] To create a sample containing a large percentage of thermaldegradation products, the 5′ fluorescein-labeled oligonucleotide34-078-01 (SEQ ID NO: 86) (200 pmole) was incubated in 100 μl 10 mMNaCO₃ (pH 10.6), 50 mM NaCl at 95° C. for 13 hours. To preventevaporation, the reaction mixture was overlaid with 60 μl ChillOut™ 14liquid wax. The reaction mixture was then divided into two equalaliquots (A and B). Aliquot A was mixed with one-tenth volume 3M NaOAcfollowed by three volumes ethanol and stored at −20° C. Aliquot B wasdephosphorylated by the addition of 0.5 μl of 1M MgCl₂ and 1 μl of1unit/μl Calf Intestine Alkaline Phosphatase (CIAP) (Promega), withincubation at 37° C. for 30 minutes. An equal volume ofphenol:chloroform:isomayl alcohol (24:24:1) was added to the samplefollowed by vortexing for one minute and then centrifugation 5 minutesat maximum speed in a microcentrifuge to separate the phases. Theaqueous phase was removed to a new tube to which one-tenth volume 3MNaOAc, and three volumes ethanol was added followed by storage at −20°C. for 30 minutes. Both aliquots (A and B) were then centrifuged for 10minutes at maximum speed in a microcentrifuge to pellet the DNA. Thepellets were then washed two times each with 80% ethanol and thendesiccated to dryness. The dried pellets were then dissolved in 70 μlddH₂O each.

[0979] The TdT reactions were conducted as follows. Six mixes wereassembled, all mixes contained 10 mM TrisOAc (pH 7.5), 10 mM MgOAc, 50mM KCl, and 2 mM dATP. Mixes 1 and 2 contained one pmole of untreated34-078-01 (SEQ ID NO: 86), mixes 3 and 4 contained 2 μl of aliquot A(above), mixes 5 and 6 contained 2 μl of aliquot B (above). To each 9 μlof mixes 1, 3 and 5, 1 μl ddH₂O was added, to each 9 μl of mixes 2, 4,and 6, 1 μl of 20 units/μl TdT (Promega) was added. The mixes wereincubated at 37° C. for 1 hour and then the reaction was terminated bythe addition of 5 μl 95% formamide with 10 mM EDTA and 0.05% markerdyes. Five microliters of each mixture was resolved by electrophoresisthrough a 20% denaturing acrylamide gel (19:1 cross-linked) with 7 Murea, in a buffer containing 45 mM Tris-Borate (pH 8.3), 1.4 mM EDTA,and imaged using with the FMBIO Image Analyzer with a 505 nm filter. Theresulting imager scan is shown in FIG. 66.

[0980] In FIG. 66, lanes 1, 3 and 5 contain untreated 34-078-01 (SEQ IDNO: 86), heat-degraded 34-078-01, and heat-degraded, dephosphorylated,34-078-01, respectively incubated in the absence of TdT. Lanes 2, 4 and6 contain, untreated 34-078-01, heat-degraded 34-078-01, andheat-degraded, dephosphorylated, 34-078-01, respectively incubated inthe presence of TdT.

[0981] As shown in FIG. 66, lane 4, TdT was unable to extend thermaldegradation products that contain a 3′-end phosphate group, andselectively extends molecules that have a 3′-end hydroxyl group.

Example 30

[0982] Specific TdT Tailing of the Products of INVADER-Directed Cleavagewith Subsequent Capture and Detection on Nitrocellulose Supports

[0983] When TdT is used to extend the specific products of cleavage, onemeans of detecting the tailed products is to selectively capture theextension products on a solid support before visualization. This Exampledemonstrates that the cleavage products can be selectively tailed by theuse of TdT and deoxynucleotide triphosphates, and that the tailedproducts can be visualized by capture using a complementaryoligonucleotide bound to a nitrocellulose support.

[0984] To extend the cleavage product produced in an INVADER-directedcleavage reaction, the following experiment was performed. Threereaction mixtures were assembled, each in a buffer of 10 mM MES (pH6.5), 0.5% Tween-20, 0.5% NP-40. The first mixture contained 5 fmols oftarget DNA-M13mp18, 10 pmols of probe oligo 32-161-2 (SEQ ID NO: 87;this probe oligonucleotide contains 3′ ddC and a Cy3 amidite group nearthe 3′ end), and 5 pmols of INVADER oligonucleotide 32-161-1 (SEQ ID NO:88; this oligo contains a 3′ ddC). The second mixture contained theprobe and INVADER oligonucleotides without target DNA. The third mixturewas the same as the first mixture, and contained the same probesequence, but with a 5′ fluorescein label (oligo 32-161-4 [SEQ ID NO:89; this oligo contains a 3′ ddC., 5′ fluorescein label, and a Cy3 dyegroup near the 3′ end]), so that the INVADER-directed cleavage productscould be detected before and after cleavage by fluorescence imaging. Theprobe only control sample contained 10 pmols of oligo 32-161-2 (SEQ IDNO: 87). Each 3 μl of enzyme mix contained 5 ng of CLEAVASE DN nucleasein 7.5 mM MgCl₂. The TdT mixture (per each 4 μl) contained: 10U of TdT(Promega), 1 mM CoCl₂, 50 mM KCl, and 100 μM of dTTP. The INVADERcleavage reaction mixtures described above were assembled in thin walltubes, and the reactions were initiated by the addition of 3 μl ofCLEAVASE DN enzyme mix. The reactions were incubated at 65° C. for 20min. After cooling to 37° C., 4 μl of the TdT mix was added and thesamples were incubated for 4 min at 37° C., Biotin-16-dUTP was thenadded to 100 μM and the samples were incubated for 50 min at 37° C. Thereactions were terminated by the addition of 1 μl of 0.5 M EDTA.

[0985] To test the efficiency of tailing the products were run on anacrylamide gel. Four microliters of each reaction mixture was mixed with2.6 μl of 95% formamide, 10 mM EDTA and 0.05% methyl violet and heatedto 90° C. for 1 min, and 3 μl were loaded on a 20% denaturing acrylamidegel (19:1 cross-linked ) with 7 M urea, in buffer containing 45 mMTris-Borate (pH 8.3), 1.4 mM EDTA. A marker (ΦX174-HinfI [fluoresceinlabeled]) also was loaded. After electrophoresis, the gel was analyzedusing a FMBIO-100 Image Analyzer (Hitachi) equipped with a 505 nmfilter. The resulting scan is shown in FIG. 67.

[0986] In FIG. 67, lane 1 contained the probe 32-161-2 only, without anytreatment. Lanes 2 and 3 contained the products of reactions run withouttarget DNA, without or with subsequent TdT tailing, respectively. Lanes4 and 5 contained the products of reactions run with target DNA, probeoligo 32-161-2 (SEQ ID NO: 87) and INVADER oligo 32-161-1 (SEQ ID NO:88), without or with subsequent TdT tailing, respectively. Lanes 6 and 7show the products of reactions containing target DNA, probe oligo32-161-4 (SEQ ID NO: 89) and INVADER oligo 32-161-1 (SEQ ID NO: 88),without or with subsequent TdT tailing, respectively. Lane M containsthe marker ΦX174-HinfI.

[0987] The reaction products in lanes 4 and 5 are the same as those seenin lanes 6 and 7, except that the absence of a 5′ fluorescein on theprobe prevents detection of the relased 5′ product (indicated as “A”near the bottom of the gel) or the TdT extended 5′ product (indicated as“B”, near the top of the gel). The Cy3-labeled 3′ portion of the cleavedprobe is visible in all of these reactions (indicated as “C”, just belowthe center of the gel).

[0988] To demonstrate detection of target-dependent INVADER-directedcleavage products on a solid support, the reactions from lanes 3 and 5were tested on the Universal GENECOMB (Bio-Rad), which is a standardnitrocellulose matrix on a rigid nylon backing styled in a comb format,as depicted in FIG. 68. Following the manufacturer's protocol, with onemodification: 10 μl of the INVADER-directed cleavage reactions were usedinstead the recommended 10% of a PCR. To capture the cleavage products,2.5 pmols of the capture oligo 59-28-1 (SEQ ID NO: 90) were spotted oneach tooth. The capture and visualization steps were conducted accordingto the manufacturer's directions. The results are shown in FIG. 68.

[0989] In FIG. 68, teeth numbered 6 and 7 show the capture results ofreactions performed without and with target DNA present. Tooth 8 showsthe kit positive control.

[0990] The darkness of the spot seen on tooth 7, when compared to tooth6, clearly indicates that products of INVADER-directed cleavage assaysmay be specifically detected on solid supports. While the UniversalGENECOMB was used to demonstrate solid support capture in this instance,other support capture methods known to those skilled in the art would beequally suitable. For example, beads or the surfaces of reaction vesselsmay easily be coated with capture oligonucleotides so that they can thenbe used in this step. Alternatively, similar solid supports may easilybe coated with streptavidin or antibodies for the capture of biotin- orhapten-tagged products of the cleavage/tailing reaction. In any of theseembodiments, the products may be appropriately visualized by detectingthe resulting fluorescence, chemiluminescence, colorimetric changes,radioactive emissions, optical density change or any otherdistinguishable feature of the product.

Example 31

[0991] Comparison of the Effects of Invasion Length and 5′ Label of theProbe on INVADER-Directed Cleavage by the CLEAVASE A/G and Pfu FEN-1Nucleases

[0992] To investigate the effect of the length of invasion as well asthe effect of the type of dye on ability of Pfu FEN-1 and the CLEAVASEA/G nuclease to cleave 5′ arms, the following experiment was performed.Three probes of similar sequences labeled with either fluorescein, TET,or Cy3, were assembled in reactions with three INVADER oligonucleotidesthat created overlapping target hybridization regions of eight, five,and three bases along the target nucleic acid, M13mp18.

[0993] The reactions were conducted as follows. All conditions wereperformed in duplicate. Enzyme mixes for Pfu FEN-1 and the CLEAVASE A/Gnuclease were assembled. Each 2 μl of the Pfu FEN-1 mix contained 100 ngof Pfu FEN-1 (prepared as described in Ex. 28) and 7.5 mM MgCl₂. Each 2μl of the CLEAVASE A/G mix contained 5.3 ng of the CLEAVASE A/G nucleaseand 4.0 mM MnCl₂. Six master mixes containing buffer, M13mp18, andINVADER oligonucleotides were assembled. Each 7 μl of mixes 1-3contained 1 fmol M13mp18, 10 pmoles INVADER oligonucleotide (34-078-4[SEQ ID NO: 39], 24-181-2 [SEQ ID NO: 91], or 24-181-1 [SEQ ID NO: 92],in 10 mM MOPS (pH 7.5), 150 mM LiCl. Each 7 μl of mixes 4-6 contained 1fmol of M13mp18, 10 pmoles of INVADER oligonucleotide [34-078-4 (SEQ IDNO: 39), 24-181-2 (SEQ ID NO: 91), or 24-181-1 (SEQ ID NO: 92)] in 10 mMTris (pH 8.0). Mixtures 1-6 were then divided into three mixtures each,to which was added either the fluorescein-labeled probe (oligo34-078-01; SEQ ID NO: 86), the Cy3-labeled probe (oligo 43-20; SEQ IDNO: 93) or the TET-labeled probe (oligo 90; SEQ ID NO: 32 containing a5′ TET label). Each 7 μl of all mixtures contained 10 pmoles ofcorresponding probe. The DNA solutions described above were covered with10 μl of CHILLOUT evaporation barrier and brought to 68° C.

[0994] The reactions made from mixes 1-3 were started with 2 μl of theCLEAVASE A/G nuclease mix, and the reactions made from mixes 4-6 werestarted with 2 μg of the Pfu FEN-1 mix. After 30 minutes at 68° C., thereactions were terminated by the addition of 8 μl of 95% formamide with10 mM EDTA and 0.05% marker dyes. Samples were heated to 90° C. for 1minute immediately before electrophoresis through a 20% denaturingacrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing45 mM Tris-Borate (pH 8.3), 1.4 mM EDTA. The products of the cleavagereactions were visualized following electrophoresis by the use of aHitachi FMBIO fluorescence imager. Results from the fluorescein-labeledprobe are shown in FIG. 69, results from the Cy3-labeled probe in FIG.70, and results from the TET-labeled probe in FIG. 71. In each of theseFigures, the products of cleavage by CLEAVASE A/G are shown in lanes 1-6and the products of cleavage by PfuFEN-1 are shown in lanes 7-12. Ineach in case the uncut material appears as a very dark band near the topof the gel, indicated by a “U” on the left. The products of cleavagedirected by INVADER oligonucleotides with 8, 5 or 3 bases of overlap(i.e., the “X” region was 8, 5, or 3 nt long) are shown in the first,second and third pair of lanes in each set, respectively and thereleased labeled 5′ ends from these reactions are indicated by thenumbers 8, 5, and 3 on the left. Note that in the cleavage reactionsshown in FIG. 70 the presence of the positively charged Cy3 dye causesthe shorter products to migrate more slowly than the larger products.These products do not contain any additional positive charges (e.g.,amino modifications as used in Example 23), and thus still carry a netnegative charge, and migrate towards the positive electrode in astandard electrophoresis run.

[0995] It can be seen from these data that the CLEAVASE A/G and PfuFEN-1 structure-specific nucleases respond differently to both dyeidentity and to the size of the piece to be cleaved from the probe. ThePfu FEN-1 nuclease showed much less variability in response to dyeidentity than did the CLEAVASE A/G nuclease, showing that any dye woldbe suitable for use with this enzyme. In contrast, the amount ofcleavage catalyzed by the CLEAVASE A/G nuclease varied substantiallywith dye identity. Use of the fluorescein dye gave results very close tothose seen with the Pfu FEN-1 nuclease, while the use of either Cy3 orTET gave dramatically reduced signal when compared to the Pfu FEN-1reactions. The one exception to this was in the cleavage of the 3 ntproduct carrying a TET dye (lanes 5 and 6, FIG. 71), in which theCLEAVASE A/G nuclease gave cleavage at the same rate as the Pfu FEN-1nuclease. These data indicate that, while CLEAVASE A/G may be used tocleave probes labeled with these other dyes, the Pfu FEN-1 nuclease is apreferred nuclease for cleavage of Cy3- and TET-labeled probes.

Example 32

[0996] Examination of the Effects of a 5′ Positive Charge on the Rate ofInvasive Cleavage Using the CLEAVASE A/G or Pfu FEN-1 Nucleases

[0997] To investigate whether the positive charges on 5′ end of probeoligonucleotides containing a positively charged adduct(s) (i.e., chargereversal technology or CRT probes as described in Ex. 23 and 24 have aneffect on the ability of the CLEAVASE A/G or Pfu FEN-1 nucleases tocleave the 5′ arm of the probe, the following experiment was performed.

[0998] Two probe oligonucleotides having the following sequences wereutilized in INVADER reactions: Probe 34-180-1:(N-Cy3)T_(NH2)T_(NH2)CCAGAGCCTAATTTGCC AGT(N-fluorescein)A, where Nrepresents a spacer containing either the Cy3 or fluorescein group (SEQID NO: 94) and Probe 34-180-2: 5′ -(N-TET)TTCCAGAGCCTAATTTGCCAGT-(N-fluorescein)A, where N represents a spacer containingeither the TET or fluorescein group (SEQ ID NO: 95). Probe 34-180-1 hasamino-modifiers on the two 5′ end T residues and a Cy3 label on the 5′end, creating extra positive charges on the 5′ end. Probe 34-180-2 has aTET label on the 5′ end, with no extra positive charges. The fluoresceinlabel on the 3′ end of probe 34-180-1 enables the visualization of the3′ cleaved products and uncleaved probes together on an acrylamide gelrun in the standard direction (i.e., with the DNA migrating toward thepositive electrode). The 5′ cleaved product of probe 34-180-1 has a netpositive charge and will not migrate in the same direction as theuncleaved probe, and is thus visualized by resolution on a gel run inthe opposite direction (i.e.; with this DNA migrating toward thenegative electrode).

[0999] The cleavage reactions were conducted as follows. All conditionswere performed in duplicate. Enzyme mixes for the Pfu FEN-1 and CLEAVASEA/G nucleases were assembled. Each 2 μl of the Pfu FEN-1 mix contained100 ng of Pfu FEN-1 (prepared as described in Ex. 28) and 7.5 mM MgCl₂.Each 2 μl of the CLEAVASE A/G nuclease mix contained 26.5 ng of CLEAVASEA/G nuclease and 4.0 mM MnCl₂. Four master mixes containing buffer,M13mp18, and INVADER oligonucleotides were assembled. Each 7 μl of mix 1contained 5 fmol M13mp18, 10 pmoles INVADER oligonucleotide 123 (SEQ IDNO: 96) in 10 mM HEPES (pH 7.2). Each 7 μl of mix 2 contained 1 fmolM13mp18, 10 pmoles INVADER oligonucleotide 123 in 10 mM HEPES (pH 7.2).Each 7 μl of mix 3 contained 5 fmol M13mp18, 10 pmoles INVADERoligonucleotide 123 in 10 mM HEPES (pH 7.2), 250 mM KGlu. Each 7 μl ofmix 4 contained 1 fmol M13mp18, 10 pmoles INVADER oligonucleotide 123 in10 mM HEPES (pH 7.2), 250 mM KGlu. For every 7 μl of each mix, 10 pmolesof either probe 34-180-1 (SEQ ID NO: 94) or probe 34-180-2 (SEQ ID NO:95) was added. The DNA solutions described above were covered with 10 μlof CHILLOUT evaporation barrier and brought to 65° C. The reactions madefrom mixes 1-2 were started by the addition of 2 μl of the Pfu FEN-1mix, and the reactions made from mixes 3-4 were started by the additionof 2 μl of the CLEAVASE A/G nuclease mix. After 30 minutes at 65° C.,the reactions were terminated by the addition of 8 μl of 95% formamidecontaining 10 mM EDTA. Samples were heated to 90° C. for 1 minuteimmediately before electrophoresis through a 20% denaturing acrylamidegel (19:1 cross-linked) with 7 M urea, in a buffer containing 45 mMTris-Borate (pH 8.3), 1.4 mM EDTA and a 20% native acrylamide gel (29:1cross-linked) in a buffer containing 45 mM Tris-Borate (pH 8.3), 1.4 mMEDTA.

[1000] The products of the cleavage reactions were visualized followingelectrophoresis by the use of a Hitachi FMBIO fluorescence imager. Theresulting images are shown in FIG. 72. FIG. 72A shows the denaturinggel, which was run in the standard electrophoresis direction, and FIG.72B shows the native gel, which was run in the reverse direction. Thereaction products produced by Pfu FEN-1 and CLEAVASE A/G nucleases areshown in lanes 1-8 and 9-16, respectively. The products from the 5 fmolM13mp18 and 1 fmol M13mp18 reactions are shown in lanes 1-4, 9-12 (5fmol) and 5-8, 13-16 (1 fmol). Probe 34-180-1 is in lanes 1-2, 5-6,9-10, 13-14 and probe 34-180-2 is in lanes 3-4, 7-8, 11-12, 15-16.

[1001] The fluorescein-labeled 3′ end fragments from all cleavagereactions are shown in FIG. 72A, indicated by a “3′” mark at the left.The 3 nt 5′ TET-labeled products are not visible in this Figure, whilethe 5′ Cy3-labeled products are shown in FIG. 72B.

[1002] The 3′ end bands in FIG. 72A can be used to compare the rates ofcleavage by the different enzymes in the presence of the different 5′end labels. It can be seen from this band that regardless of the amountof target nucleic acid present, both the Pfu FEN-1 and the CLEAVASE A/Gnucleases show more product from the 5′ TET-labeled probe. With the PfuFEN-1 nuclease this preference is modest, with only an approximately 25to 40% increase in signal. In the case of the CLEAVASE A/G nuclease,however, there is a strong preference for the 5′ TET label. Therefore,although when the charge reversal method is used to resolve theproducts, a substantial amount of product is observed from the CLEAVASEA/G nuclease-catalyzed reactions, the Pfu FEN-1 nuclease is a preferredenzyme for cleavage of Cy3-labeled probes.

Example 33

[1003] The Use of Universal Bases in the Detection of Mismatches byINVADER Directed Cleavage

[1004] The term “degenerate base” refers to a base on a nucleotide thatdoes not hydrogen bond in a standard “Watson-Crick” fashion to aspecific base complement (i.e., A to T and G to C). For example, theinosine base can be made to pair via one or two hydrogen bonds to all ofthe natural bases (the “wobble” effect) and thus is called degenerate.Alternatively, a degenerate base may not pair at all; this type of basehas been referred to as a “universal” base because it can be placedopposite any nucleotide in a duplex and, while it cannot contributestability by base-pairing, it does not actively destabilize by crowdingthe opposite base. Duplexes using these universal bases are stabilizedby stacking interactions only. Two examples of universal bases,3-nitropyrrole and 5-nitroindole, are shown in FIG. 73. Inhybridization, placement of a 3-nitropyrrole three bases from a mismatchposition enhances the differential recognition of one base mismatches.The enhanced discrimination seems to come from the destabilizing effectof the unnatural base (i.e., an altered T_(m) in close proximity to themismatch). To test this same principle as a way of sensitively detectingmismatches using the INVADER-directed cleavage assay, INVADERoligonucleotides were designed using the universal bases shown in FIG.73, in the presence or absence of a natural mismatch. In theseexperiments, the use of single nitropyrrole bases or pairs ofnitroindole bases that flank the site of the mismatch were examined.

[1005] The target, probe and INVADER oligonucleotides used in theseassays are shown in FIG. 74. A 43 nucleotide oligonucleotide (oligo 109;SEQ ID NO: 97) was used as the target. The probe oligonucleotide (oligo61; SEQ ID NO: 50) releases a net positively charged labeled productupon cleavage. In FIG. 74, the INVADER oligonucleotide is shownschematically above the target oligonucleotide as an arrow; the largearrowhead indicates the location of the mismatch between the INVADERoligos and the target. Under the target oligonucleotide, the completelycomplementary, all natural (i.e., no universal bases) INVADER oligo(oligo 67; SEQ ID NO: 5 1) and a composite of INVADER oligos containinguniversal bases (“X”) on either side of the mismatch (“M”) are shown.The following INVADER oligos were employed: oligo 114 (SEQ ID NO: 98),which contains a single nt mismatch; oligo 115 (SEQ ID NO: 99), whichcontains two 5-nitroindole bases and no mismatch; oligo 116 (SEQ ID NO:100), which contains two 5-nitroindole bases and a single nt mismatch;oligo 112 (SEQ ID NO: 101), which contains one 3-nitropyrrole base andno mismatch; oligo 113 (SEQ ID NO: 102), which contains one5-nitropyrrole base and a single nt mismatch; and oligo 67 (SEQ ID NO: 51), which is completely complementary to the target.

[1006] The INVADER-directed cleavage reactions were carried out in 10 μlof 10 mM MOPS (pH 7.2), 100 mM KCl, containing 1 μM of the appropriateinvading oligonucleotide (oligos 67, 112-116), 10 nM synthetic target109, 1 μM Cy-3 labeled probe 61 and 2 units of CLEAVASE DV (prepared asdescribed in Ex. 27). The reactions were overlayed with Chill-Out®liquid wax, brought to the appropriate reaction temperature, 52° C., 55°C., or 58° C. and initiated with the addition of 1 μl of 40 mM MnCl₂.Reactions were allowed to proceed for 1 hour and were stopped by theaddition of 10 μl formamide. One fourth of the total volume of eachreaction was loaded onto 20% non-denaturing polyacrylamide gels, whichwere electrophoresed in the reverse direction. The products werevisualized using an Hitachi FMBIO-100 fluorescent scanner using a 585 nmfilter. The resulting images are shown in FIGS. 75A-C. In each panel,lanes 1-6 contain reactions products from reactions using INVADER oligo67, 114, 115, 116, 112 and 113, respectively. Reactions run at 52° C.,55° C. and 58° C. are shown in Panels A, B and C., respectively.

[1007] These data show that two flanking 5-nitroindoles display asignificantly greater differentiation then does the one 3-nitropyrrolesystem, or the all natural base hybridization, and this increasedsensitivity is not temperature dependent. This demonstrates that the useof universal bases is a useful means of sensitively detecting singlebase mismatches between the target nucleic acid and the complex ofdetection oligonucleotides of the present invention.

Example 34

[1008] Detection of Point Mutations in the Human Ras Oncogene Using aMiniprobe

[1009] It is demonstrated herein that very short probes can be used forsensitive detection of target nucleic acid sequences (Ex. 37). In thisExample, it is demonstrated that the short probes work very poorly whenmismatched to the target, and thus can be used to distinguish a givennucleic acid sequence from a close relative with only a single basedifference. To test this system synthetic human ras oncogene targetsequences were created that varied from each other at one position.Oligonucleotide 166 (SEQ ID NO: 103) provided the wild-type ras targetsequence. Oligonucleotide 165 (SEQ ID NO: 104) provided the mutant rastarget sequence. The sequence of these oligonucleotides are shown inFIG. 76, and the site of the sequence variation in the sitecorresponding to codon 13 of the ras gene is indicated. The INVADERoligonucleotide (oligo 162) has the sequence:5′-G_(s)C_(s)T_(s)C_(s)A_(s)A_(s)G_(s)G_(s)C_(s)ACTCTTGCC TACGA-3′ (SEQID NO: 105), where the “S” indicates thiol linkages (i.e., these are2′-deoxynucleotide-5′-O-(1-thiomonophates)). The miniprobe (oligo 161)has the sequence: 5′-(N-Cy3) T_(NH2)T_(NH2)CACCAG-3′ (SEQ ID NO: 106)and is designed to detect the mutant ras target sequence (i.e., it iscompletely complementary to oligo 165). The stacker oligonucleotide(oligo 164) has the sequence:5′-C_(s)T_(s)C_(s)C_(s)A_(s)A_(s)C_(s)T_(s)A_(s)CCACAAGTTTATATTCAG-3′(SEQ ID NO: 107). A schematic showing the assembly of theseoligonucleotides into a cleavage structure is depicted in FIG. 76.

[1010] Each cleavage reaction contained 100 nM of both the invading(oligo 162) and stacking (oligo 164) oligonucleotides, 10 μM Cy3-labeledprobe (oligo 161) and 100 pM of either oligo 165 or oligo 166 (targetDNA) in 10 μl of 10 mM HEPES (pH 7.2), 250 mM KGlu, 4 mM MnCl₂. The DNAmixtures were overlaid with mineral oil, heated to 90° C. for 15 secthen brought to a reaction temperature of 47°, 50°, 53° or 56° C.Reactions were initiated by the addition of 1 μl of 100 ng/μl Pfu FEN-1.Reactions were allowed to proceed for 3 hours and stopped by theaddition of 10 μl formamide. One fourth of the total volume od eachreaction was loaded onto a 20% non-denaturing polyacrylamide gel, whichwas electrophoresed in the reverse direction. The gel was scanned usingan Hitachi FMBIO-100 fluorescent scanner fitted with a 585 nm filter,and the resulting image is shown in FIG. 77.

[1011] In FIG. 77, for each reaction temperature tested, the productsfrom reactions containing either the mutant ras target sequence (oligo165) or the wild-type (oligo 166) are shown.

[1012] These data demonstrate that the miniprobe can be used tosensitively discriminate between sequences that differ by a singlenucleotide. The miniprobe was cleaved to produce a strong signal in thepresence of the mutant target sequence, but little or no miniprobe wascleaved in the presence of the wild-type target sequence. Furthermore,the discrimination between closely related targets is effective over atemperature range of at least 10° C., which is a much broader range oftemperature than can usually be tolerated when the selection is based onhybridization alone (e.g., hybridization with ASOs). This suggests thatthe enzyme may be a factor in the discrimination, with the perfectlymatched miniprobe being the preferred substrate when compared to themismatched miniprobe. Thus, this system provides sensitive and specificdetection of target nucleic acid sequences.

Example 35

[1013] Effects of 3′ End Identity on Site of Cleavage of A ModelOligonucleotide Structure

[1014] As described in the Examples above, structure-specific nucleasescleave near the junction between single-stranded and base-paired regionsin a bifurcated duplex, usually about one base pair into the base-pairedregion. It was shown in Example 10 that thermostable 5′ nucleases,including those of the present invention (e.g., CLEAVASE BN nuclease,CLEAVASE A/G nuclease), have the ability to cleave a greater distanceinto the base paired region when provided with an upstreamoligonucleotide bearing a 3′ region that is homologous to a 5′ region ofthe subject duplex, as shown in FIG. 26. It has also been determinedthat the 3′ terminal nucleotide of the INVADER oligonucleotide may beunpaired to the target nucleic acid, and still shift cleavage the samedistance into the down stream duplex as when paired. It is shown in thisExample that it is the base component of the nucleotide, not the sugaror phosphate, that is necessary to shift cleavage.

[1015]FIGS. 78A and B shows a synthetic oligonucleotide that wasdesigned to fold upon itself, and that consists of the followingsequence: 5′-GTTCTCTGCTCTCTGGTCGCTGTCTCGCTTGTGAAACAAGCGAGACAGCGTGGTCTCTCG-3′ (SEQ ID NO: 29). Thisoligonucleotide is referred to as the “S-60 Hairpin.” The 15 basepairhairpin formed by this oligonucleotide is further stabilized by a“tri-loop” sequence in the loop end (i.e., three nucleotides form theloop portion of the hairpin) (Hiraro et al., Nucleic Acids Res., 22(4):576 [1994]). FIG. 78B shows the sequence of the P-15 oligonucleotide(SEQ ID NO: 30) and the location of the region of complementarity sharedby the P-15 and S-60 hairpin oligonucleotides. In addition to the P-15oligonucleotide shown, cleavage was also tested in the presence of theP-14 oligonucleotide (SEQ ID NO: 108) (P-14 is one base shorter on the3′ end as compared to P-15), the P-14 with an abasic sugar (P-14d; SEQID NO: 109) and the P14 with an abasic sugar with a 3′ phosphate (P-14dp; SEQ ID NO: 110). A P-15 oligo with a 3′ phosphate, P-15p (SEQ ID NO:111) was also examined. The black arrows shown in FIG. 78 indicate thesites of cleavage of the S-60 hairpin in the absence (top structure; A)or presence (bottom structure; B) of the P-15 oligonucleotide.

[1016] The S-60 hairpin molecule was labeled on its 5′ end withfluorescein for subsequent detection. The S-60 hairpin was incubated inthe presence of a thermostable 5′ nuclease in the presence or theabsence of the P-15 oligonucleotide. The presence of the full duplexthat can be formed by the S-60 hairpin is demonstrated by cleavage withthe CLEAVASE BN 5′ nuclease, in a primer-independent fashion (i.e., inthe absence of the P-15 oligonucleotide). The release of 18 and19-nucleotide fragments from the 5′ end of the S-60 hairpin moleculeshowed that the cleavage occurred near the junction between the singleand double stranded regions when nothing is hybridized to the 3′ arm ofthe S-60 hairpin (FIG. 27, lane 2).

[1017] The reactions shown in FIG. 78C were conducted in 10 μl 1×CFLPbuffer with 1 mM MnCl₂ and 50 mM K-Glutamate, in the presence of 0.02 μMS-60, 0.5 μM INVADER oligonucleotide and 0.01 ng per μl CLEAVASE BNnuclease. Reactions were incubated at 40° C. for 5 minutes and stoppedby the addition of 8 μl of stop buffer (95% formamide, 20 mM EDTA, 0.02%methyl violet). Samples were heated to 75° C. for 2 min immediatelybefore electrophoresis through a 15% acrylamide gel (19:1 cross-linked),with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.Gels were then analyzed with a FMBIO-100 Image Analyzer (Hitachi)equipped with 505 nm filter. The resulting image is shown in FIG. 78C.

[1018] In FIG. 78C lane 1 contains products from the no enzyme control;lane 2 contains products from a reaction run in the absence of anINVADER oligo; lanes 3-6 contain products from reactions run thepresence of the P-14d, P-14dp, P-15 and P-15p INVADER oligos,respectively.

[1019] From the data shown in FIG. 78C., it can be seen that the use ofthe P-15 INVADER oligonucleotide produces a shift in the cleavage site,while the P14 INVADER oligonucleotide with either a ribose (P14d) or aphosphorylated ribose (P14dp) did not This indicates that the 15thresidue of the INVADER oligonucleotide must have the base group attachedto promote the shift in cleavage. Interestingly, the addition ofphosphate to the 3′ end of the P15 oligonucleotide apparently reversedthe shifting of cleavage site. The cleavage in this lane may in fact becleavage in the absence of an INVADER oligonucleotide as is seen in lane2. In experiments with 5′ dye-labeled INVADER oligonucleotides with 3′phosphate groups these oligonucleotides have been severely retarded ingel migration, suggesting that either the enzyme or another constituentof the reaction (e.g., BSA) is able to bind the 3′ phosphateirrespective of the rest of the cleavage structure. If the INVADERoligonucleotides are indeed being sequestered away from the cleavagestructure, the resulting cleavage of the S-60 hairpin would occur in a“primer-independent” fashion, and would thus not be shifted.

[1020] In addition to the study cited above, the effects of othersubstituents on the 3′ ends of the INVADER oligonucleotides wereinvestigated in the presence of several different enzymes, and in thepresence of either Mn++ or Mg++. The effects of these 3′ endmodifications on the generation of cleaved product are summarized in thefollowing table. All of modifications were made during standardoligonucleotide synthesis by the use of controlled pore glass (CPG)synthesis columns with the listed chemical moiety provided on thesupport as the synthesis starting residue. All of these CPG materialswere obtained from Glen Research Corp. (Sterling, Va.).

[1021]FIG. 79 provides the structures for the 3′ end substituents usedin these experiments. TABLE 4 Modification Studies At 3' End Of INVADEROligo Extension Effect on INVADER Rxn. (As By Terminal INVADER)Enzyme:Condition - 3'-End Modification Trasferase Effect 3' phosphate noA:5 - inhibits reaction, Glen part # 20-2900-42 no detectable activity3' acridine yes, poorly A:5 - decrease in activity, <10% Glen part #20-2973-42 B:5 - decrease in activity, <10% B:4 - decrease in activity,<10% C:1 -decrease in activity, <10% C:2 - decrease in activity, ˜20%C:4 - decrease in activity, ˜50% C:3 - decrease in activity, <5% 3'carboxylate no A:1 - decrease in activity, ˜50% Glen part # 20-4090-42activity shift in cleavage site C:3 - reduces rate, <10% activity 3'nitropyrole yes A:5 - increase in activity, ˜2X Glen part # 20-2143-423' nitroindole yes A:5 - decrease in activity, ˜33% Glen part #20-2144-42 activity 3' arabinose yes A:5 - decrease in activity, ˜50%Glen part # 10-4010-90 activity 3'dideoxyUTP- no A:5 - decrease inactivity, ˜40% flourescein activity 3'-3'linkage no A:1 - equivalentcleavage Glen part # 20-0002-01 activity shift in cleavage site C:3 -decrease in activity, ˜25% activity 3' glyceryl yes, very C:3 - decreasein activity, ˜30% Glen part # 20-2902-42 poorly activity loss ofspecificity of cleavage (2 sites) 3' amino modifier C7 yes C:3 -decrease in activity, ˜30% Glen part # 20-2957-42 activity loss ofspecificity, multiple sites

[1022] Enzymes:

[1023] A) CLEAVASE DV nuclease

[1024] B) CLEAVASE BN nuclease

[1025] C) Pfu FEN-1

[1026] Condition:

[1027] 1) 4 mM MnCl₂, 150 mM LiCl

[1028] 2) 4 mM MnCl₂, 50 mM KCl

[1029] 3) 7.5 mM MgCl₂, no monovalent

[1030] 4) 4 mM MgCl₂, 50 mM KCl

[1031] 5) 10 mM MgOAc, 50 mM KCl

[1032] It can be seen from these data that many different modificationscan be used on the 3′ end of the INVADER oligonucleotide withoutdetriment. In various embodiments of the present invention, such 3′ endmodifications maybe used to block, facilitate, or otherwise alter thehybridization characteristics of the INVADER oligonucleotide, (e.g., toincrease discrimination against mismatches, or to increase tolerance ofmismatches, or to tighten the association between the INVADERoligonucleotide and the target nucleic acid). Some substituents may beused to alter the behavior of the enzyme in recognizing and cleavingwithin the assembled complex.

[1033] Altered 3′ ends may also be used to prevent extension of theINVADER oligonucleotide by either template-dependent ortemplate-independent nucleic acid polymerases. The use of otherwiseunmodified dideoxynucleotides (i.e., without attached dyes or othermoieties) are a particularly preferred means of blocking extension ofINVADER oligonucleotides, because they do not decrease cleavageactivity, and they are absolutely unextendable.

Example 36

[1034] Effect of Probe Concentration, Temperature and a StackerOligonucleotide on the Cleavage of Miniprobes by INVADER-DirectedCleavage

[1035] The stacker oligonucleotides employed to form cleavage structuresmay serve two purposes in the detection of a nucleic acid target using aminiprobe. The stacker oligonucleotide may help stabilize theinteraction of the miniprobe with the target nucleic acid, leading togreater accumulation of cleaved probe. In addition, the presence of thisoligo in the complex elongates the duplex downstream of the cleavagesite, which may enhance the cleavage activity of some of the enzymes ofthe present invention. An example of different preferences for thelength of this duplex by different structure-specific nucleases is seenin the comparison of the CLEAVASE BN nuclease and the Mja FEN-1 nucleasecleavage of 8 bp and 12 bp duplex regions in FIG. 65. Increased affinityof the enzyme for the cleavage structure also results in increasedaccumulation of cleaved probe during reactions done for a set amount oftime.

[1036] The amount of miniprobe binding to the target is also affected bythe concentration of the miniprobe in the reaction mixture. Even when aminiprobe is only marginally likely to hybridize (e.g., when thereaction is performed at temperatures in excess of the expected meltingtemperature of the probe/target duplex), the amount of probe on thetarget at any given time can be increased by using high concentrationsof the miniprobe.

[1037] The need for a stacker oligonucleotide to enhance cleavage of theminiprobe was examined at both low and high probe concentrations. Thereactions were carried out in 10 μl of 10 mM HEPES (pH 7.2), 250 mMKGIu, 4 mM MnCl₂, containing 100 nM of both the invading (oligo 135; SEQID NO: 112) and stacking oligonucleotides (oligo 147; SEQ ID NO: 113)and 100 pM ssM13 DNA. The reactions were overlayed with mineral oil,heated to 90° C. for 15 sec then brought to the reaction temperature.Reactions were performed at 35°, 40°, 45°, 50°, 55°, 60°, and 65° C. Thecleavage reactions were initiated by the addition of 1 μl of 100 ng/μlPfu FEN-I and 1 μl of varying concentrations of Cy-3 labeled 142miniprobe oligonucleotide (SEQ ID NO: 114). Reactions were allowed toproceed for 1 hour and stopped by the addition of 10 μl formaldehyde.One fourth of the total volume of each reaction was loaded onto 20%non-denaturing polyacrylamide gels, which were electrophoresed in thereverse direction. Gels were visualized using an Hitachi FMBIO-100fluorescent scanner using a 585 nm filter. The fluorescence in eachproduct band was measured and the graph shown in FIG. 80 was createdusing a Microsoft Excel spreadsheet.

[1038] The data summarized in FIG. 80 showed that the concentration ofthe miniprobe had a significant effect on the final measure of product,showing dramatic increases as the concentration was raised. Increases inthe concentration of the miniprobe also shifted the optimum reactiontemperature upward. It is known in the art that the concentration of thecomplementary strands in a hybridization will affect the apparent T_(m)of the duplex formed between them. More significantly to the methods andcompositions of the present invention is the fact that the presence ofthe stacker oligonucleotide has a profound influence on the cleavagerate of the miniprobe at all probe concentrations. At each of the probeconcentrations the presence of the stacker as much as doubled the signalfrom the cleavage product. This demonstrated the utility of using thestacker oligonucleotide in combination with the miniprobes describedherein.

Example 37

[1039] The Presence of a Mismatch in the INVADER OligonucleotideDecreases the Cleavage Activity of the CLEAVASE A/G Nuclease

[1040] In any nucleic acid detection assay it is of additional benefitif the assay can be made to sensitively detect minor differences betweenrelated nucleic acids. In the following experiment, model cleavagesubstrates were used that were identical except for the presence orabsence of a mismatch near the 3′ end of the INVADER oligonucleotidewhen hybridized to the model target nucleic acid. The effect of amismatch in this region on the accumulation of cleaved probe was thenassessed.

[1041] To demonstrate the effect of the presence of a mismatch in theINVADER oligonucleotide on the ability of the CLEAVASE A/G nuclease tocleave the probe oligonucleotide in an INVADER assay the followingexperiment was conducted. Cleavage of the test oligonucleotide IT-2 (SEQID NO: 115) in the presence of INVADER oligonucleotides IT-1 (SEQ ID NO:116) and IT-1A4 (SEQ ID NO: 117). Oligonucleotide IT-1 is fullycomplementary to the 3′ arm of IT-2, whereas oligonucleotide IT-1A4 hasa T->A substitution at position 4 from the 3′ end that results in an A/Amismatch in the INVADER-target duplex. Both the matched and mismatchedINVADER oligonucleotides would be expected to hybridize at thetemperature at which the following reaction was performed. FIG. 81provides a schematic showing IT-1 annealed to the folded IT-2 structureand showing IT-1A4 annealed to the folded IT-2 structure.

[1042] The reactions were conducted as follows. Test oligonucleotideIT-2 (0.1 μM), labeled at the 5′ end with fluorescein (Integrated DNATechnologies), was incubated with 0.26 ng/μl CLEAVASE AG in 10 μl ofCFLP® buffer with 4 mM MgCl₂, in the presence of 1 μM IT-1 or IT-1A4 at40° C. for 10 min; a no enzyme control was also run. Samples wereoverlaid with 15 μl Chill-Out® liquid wax to prevent evaporation.Reactions were stopped by addition of 4 μl stop buffer (95% formamide,20 mM EDTA, 0.02% methyl violet). The cleavage products were separatedon a 20% denaturing polyacrylamide gel and analyzed with the FMBIO-100Image Analyzer (Hitachi) equipped with 505 nm filter. The resultingimage is shown in FIG. 82.

[1043] In FIG. 82, lane 1 contains reaction products from the no enzymecontrol and shows the migration of the uncut IT-2 oligo; lanes 2-4contain products from reactions containing no INVADER oligo, the IT-1INVADER oligo and the IT-1A4 INVADER oligo, respectively.

[1044] These data show that cleavage is markedly reduced by the presenceof the mismatch, even under conditions in which the mismatch would notbe expected to disrupt hybridization. This demonstrates that the INVADERoligonucleotide binding region is one of the regions within the complexin which can be used for mismatch detection, as revealed by a drop inthe cleavage rate.

Example 38

[1045] Comparison of the Activity of the Pfu FEN-1 and Mja EN-1Nucleases in the INVADER Reaction

[1046] To compare the activity of the Pfu FEN-1 and the Mja FEN-1nucleases in INVADER reaction the following experiment was performed. Atest oligonucleotide IT3 (SEQ ID NO: 118) that forms an INVADER-Targethairpin structure and probe oligonucleotide PR1 (SEQ ID NO: 119) labeledat the 5′ end with fluorescein (Integrated DNA Technologies) wereemployed in INVADER assays using either the Pfu FEN-1 or the Mja FEN-1nucleases.

[1047] The assays were conducted as follows. Pfu FEN-1 (13 ng/μl) andMja FEN-1 (10 ng/μl) (prepared as described in Ex. 28) were incubatedwith the IT3 (0.1 nM) and PR1 (2 and 5 μM) oligonucleotides in 10 μLCFLP® buffer, 4 mM MgCl₂, 20 mg/ml tRNA at 55° C. for 41 min. Sampleswere overlaid with 15 μl Chill-Out® evaporation barrier to preventevaporation. Reactions were stopped by addition of 70 μl stop buffer(95% formamide, 20 mM EDTA, 0.02% methyl violet). Reaction products (1μ1) were separated on a 20% denaturing polyacrylamide gel, visualizedusing a fluroimager and the bands corresponding to the probe and theproduct were quantitiated. The resulting image is shown in FIG. 83. InFIG. 83, the turnover rate per target per minute is shown below theimage for each nuclease at each concentration of probe and targettested.

[1048] It was demonstrated in Example 32 that the use of the Pfu FEN-1structure-specific nuclease in the INVADER-directed cleavage reactionresulted in a faster rate of product accumulation than did the use ofthe CLEAVASE A/G. The data presented here demonstrates that the use ofMja FEN-1 nuclease with the fluorescein labeled probe further increasesthe amount of product generated by an average of about 50%,demonstrating that, in addition to the Pfu FEN-1 nuclease, the Mja FEN-1nuclease is a preferred structure-specific nuclease for the detection ofnucleic acid targets by the method of the present invention.

Example 39

[1049] Detection of RNA Target Nucleic Acids Using Miniprobe and StackerOligonucleotides

[1050] In addition to the detection of the M13 DNA target materialdescribed above, a miniprobe/stacker system was designed to detect theHCV-derived RNA sequences described in Example 19. A probe ofintermediate length, either a long mid-range or a short standard probe,was also tested. The miniprobe used (oligo 42-168-1) has the sequence:5′-TET-CCGGTCGTCCTGG-3′ (SEQ ID NO: 120), the stacker oligonucleotideused (oligo 32-085) with this miniprobe has the sequence:5′-CAATTCCGGTGTACTACCGGTTCC-3′ (SEQ ID NO: 121). The slightly longerprobe, used without a stacker (oligo 42-088), has the sequence:5′-TET-CCGGTCGTCCTGGCAA-3′ (SEQ ID NO: 122). The INVADER oligonucleotideused with both probes has the sequence: 5′-GTTTATCCAAGAAAGGACCCGGTC-3′(SEQ ID NO: 47). The reactions included 50 fmole of target RNA, 10 pmoleof the INVADER oligonucleotide and 5 pmole of the miniprobeoligonucleotide in 10 μl of buffer containing 10 mM MES, pH 6.5 with 150mM LiCl, 4 mM MnCl₂, 0.05% each Tween-20 and NP-40, and 39 units ofRNAsin (Promega). When used, 10 pmoles of the stacker oligonucleotidewas added. These components were combined, overlaid with CHILLOUTevaporation barrier, and warmed to 50° C.; the reactions were started bythe addition of 5 polymerase units of DNAPTth, to a final reactionvolume of 10 μl. After 30 minutes at 50° C., reactions were stopped bythe addition of 8 μl of 95% formamide, 10 mM EDTA and 0.02% methylviolet. The samples were heated to 90° C. for 1 minute and 2.5 μl ofeach of these reactions were resolved by electrophoresis through a 20%denaturing polyacrylamide (19:1 cross link) with 7M urea in a buffer of45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA, and the labeled reactionproducts were visualized using the FMBIO-100 Image Analyzer (Hitachi).The resulting image is shown in FIG. 84.

[1051] In FIG. 84, lanes 1 and 2 show the products of reactionscontaining the HCV INVADER oligonucleotide and the longer probe (oligo42-088), without and with the target RNA present, respectively. Lanes 3,4, and 5 show the products of reactions containing the INVADERoligonucleotide and the shorter probe (oligo 42-168-1). Lane 3 is acontrol reaction without target RNA present, while lanes 4 and 5 havethe target, but are without or with the stacker oligonucleotide,respectively.

[1052] Under these conditions the slightly longer (16 nt) probeoligonucleotide was cleaved quite easily without the help of a stackeroligonucleotide. In contrast, the shorter probe (13 nt) required thepresence of the stacker oligonucleotide to produce detectable levels ofcleavage. These data show that the miniprobe system of target detectionby INVADER-directed cleavage is equally applicable to the detection ofRNA and DNA targets. In addition, the comparison of the cleavageperformance of longer and shorter probes in the absence of a stackeroligonucleotide give one example of the distinction between theperformance of the miniprobe/stacker system and the performance of themid-range and long probes in the detection of nucleic acid targets.

Example 40

[1053] Effect of an Unpaired 3′ Tail on Transcription from a Complete(Un-Nicked) Promoter

[1054] In designing the method of transcription-based visualization ofthe products of INVADER-directed cleavage, it was first necessary toassess the effect of a 3′ tail on the efficiency of transcription from afull length promoter. The duplexes tested in this Example are shown atthe bottom of FIG. 93, and are shown schematically in FIGS. 85A-C.

[1055] Transcription reactions were performed using the MEGAshortscript™system from Ambion, Inc. (Austin, Tex.), in accordance with themanufacturer's instructions with the exception that a fluoresceinlabeled ribonucleotide was added. Each DNA sample was assembled in 4 μlof RNAse-free dH₂O. Reactions 1-3 each contained 10 pmole of the copytemplate oligo 150 (SEQ ID NO: 123); reaction 2 contained 10 pmole ofthe promoter oligo 151 (SEQ ID NO: 124); sample 3 contained 10 pmole ofthe 3′ tailed promoter oligo 073-065 (SEQ ID NO: 125); sample 4 had noadded DNA. To each sample, 6 μl of a solution containing 1 μl of 10×Transcription Buffer, 7.5 mM each rNTP, 0.125 mM fluorescein-12-UTP(Boehringer) and 1 μl T7 MEGAshortscript™ Enzyme Mix was added. Thesamples were then incubated at 37° C. for 1 hour. One microliter ofRNase-free DNase 1 (2U/μl) was added to each sample and the samples wereincubated an additional 15 minutes at 37° C. The reactions were thenstopped by the addition of 10 μl of a solution of 95% formamide, 5 mMNa₂EDTA, with loading dyes. All samples were heated to 95° C. for 2minutes and 4 μl of each sample were resolved by electrophoresis througha 20% denaturing acrylamide gel (19:1 cross-linked) with 7M urea, in abuffer containing 45 mM Tris-Borate (pH 8.3), 1.4 mM EDTA. The gel wasanalyzed with a FMBIO II fluorescence image analyzer, and the resultingimage is shown in FIG. 93. The RNA produced by successful transcriptionappears near the middle of the panel, as indicated (“RNA”).

[1056] Examination of the products of transcription shown in lanes 2 and3 show that the presence of the 3′ tail on the full-length promoter hasan adverse affect on the efficiency of transcription, but does not shutit off completely. Because the objective of the transcription-basedvisualization assays of the present invention is to discriminate betweenuncleaved probe and the shorter products of the invasive cleavage assay(cut probe), these data indicate that production of a full-lengthpromoter in the cleavage reaction would be difficult to resolve from thebackground created by transcription from promoters containing theuncleaved probe if no other oligonucleotides were included in the assay.Means of suppressing transcription from such a branched promoter arediscussed in the Description of the Invention and discussed below in Ex.43.

Example 41

[1057] Examination of the Influence of the Position of the Nick on theEfficiency of Transcription from Partial and Complete CompositeBacteriophage T7 Promoters

[1058] In the Description of the Invention, the procedure for testingprospective promoter pieces for suitability in an invasivecleavage-linked assay is described. One aspect of the test is to examinethe effect a chosen nick site has on the efficiency of transcriptionfrom the final composite promoter. In addition, the individual pieces ofnicked promoter are tested for transcription activity in the presence ofthe full-length un-nicked strand. In this experiment, a comparison onthese points is made between a composite promoter having a nick in thenon-template strand between nucleotides −11 and −10 relative to theinitiation site (+1), and a promoter having a nick on the same strand,but positioned between nucleotides −8 and −7. The Figure numbers for theschematic representations of the contents of each reaction are indicatedbelow each lane (e.g., 85A=FIG. 85A). The site where the nick would bein a fully assembled composite promoter using the reactionoligonucleotides is also indicated below each lane (“−11/−10” and“−8/−7”).

[1059] Transcription reactions were performed using the MEGAshortscript™system, in accordance with the manufacturer's instructions, but with theexception that a fluorescein labeled ribonucleotide was added. Each DNAsample was assembled in 4 μl of RNAse-free dH₂O. Reaction 1 had no addedDNA. Reactions 2-9 each contained 10 pmole of the copy template oligo150 (SEQ ID NO: 123). Reactions 3 and 4 contained 10 pmole of the −11“cut” probe (oligo 073-061-01; SEQ ID NO: 127) or 20 pmole of the −10partial promoter oligo 073-061-02 (SEQ ID NO: 130), respectively, andreaction 5 contained both. Reactions 6 and 7 contained either the 10pmole of the −8 “cut” probe (oligo 073-062-01; SEQ ID NO: 126) or 20pmoles of the −7 partial promoter oligo 073-062-02 (SEQ ID NO: 129),respectively, and reaction 8 contained them both. Reaction 9 contained10 pmole of the intact promoter oligo 151 (SEQ ID NO: 124).

[1060] The transcription reactions were initiated, incubated, terminatedand the reaction products were resolved and imaged as described in Ex.40. The resulting image is shown in FIG. 92. The reaction numberscorrespond to the lane numbers above the image. The RNA created bysuccessful transcription appears in the upper third of the image.Comparison to the positive control reaction (rxn. 9) shows that thefull-length RNA produced by each of the composite promoters is the samesize as that produced in the control reaction, indicated thattranscription initiated at the same site in each reaction.

[1061] In FIG. 92, lanes 3, 4, and 5 compare transcription from the twospecies of partially assembled promoters (see schematics in FIGS. 86Aand B) and the fully assembled composite promoter (FIG. 88B) having anick between nucleotides −11 and −10 relative to the start oftranscription. It can be seen from these data that neither partialpromoter (lanes 3 and 4) is able to support transcription of the copytemplate, but that the composite promoter (lane 5) with this nick siteis strongly transcribed. Surprisingly, comparison to the controlreaction (lane 9) shows that the presence of a nick at this site(−11/−10) actually enhances transcription. While not limiting thepresent invention to any particular mechanism, it is believed that theenhancement of transcription is a result of both suppressing theformation of the shorter abortive transcripts and by allowing greateraccumulation of the full length product. This result is highlyreproducible.

[1062] In FIG. 92, lanes 6, 7, and 8 compare transcription a similar setof partial and complete promoters in which the nick is shifted 3residues closer to the transcription start site. Examination of lane 6shows that the presence of 3 extra bases on the −8“cut” probe (comparedto the −11 “cut” probe in lane 3) allow this partial promoter toinitiate transcription. This indicates that the −8/−7 site would be apoor choice for use in this embodiment of the present invention.

[1063] This experiment demonstrates the process for determining thesuitable placement of a nick within a promoter assembly to achieve thedesired result. Similar tests can easily be designed for testing othernicks within the bacteriophage T7 promoter tested in this Example, orfor testing suitable nick placement in any desired phage, prokaryotic oreukaryotic promoter.

Example 42

[1064] Detection of the Products of INVADER-Directed Cleavage throughTranscription from a Composite Promoter

[1065] The Examples described above indicate that a smalloligonucleotide can be used to complete assembly of a composite T7promoter, thereby enabling transcription from that promoter. EarlierExamples demonstrate that the invasive cleavage reaction can be usedrelease specific small oligonucleotide products from longer probeoligonucleotides. In this Example, it is demonstrated that these twoobservations can be combined, and that the products of the invasivecleavage reaction can be used to complete a promoter and enablesubsequent transcription. The schematic representations of the compositepromoters tested in this Example are shown in FIG. 88.

[1066] Two invasive cleavage reactions were set up, one without (rxn. 1)and one with (rxn. 2) input target DNA. The reactions (1 and 2)comprised 10 mM MOPS (pH 7.5), 0.05% Tween-20, 0.05% NP-40 and 20 pmolesprobe oligo 073-067-01 (SEQ ID NO: 132) and 10 pmoles INVADER oligo073-073-02 (SEQ ID NO: 134) in a volume of 14 μl. Reaction 2 alsoincluded 100 fmoles M13mp18 ssDNA. The samples were placed at 60° C. and6 μl of a solution containing 20 ng of Mja FEN-1 and 40 mM Mg₂Cl wereadded to each sample to start the reactions. The samples were incubatedat 60° C. for 30 minutes and stopped by the addition of 3 μl of 2.5MNaOAc, 83 mM Na₂EDTA (pH 8.0). Each sample was transferred to a 1.5 mlmicrocentrifuge tube and then the DNAs were precipitated by the additionof 60 μl of chilled 100% ethanol, and were stored at −20° C. for 20minutes. The pellets were collected by microcentrifugation, washed oncewith 80% ethanol to remove excess salt, then dried under vacuum. Theproduct of this invasive cleavage reaction is a 12 nt oligonucleotidehaving the sequence:

[1067] 5′-CGAAATTAATAC-3′ (SEQ ID NO: 128), termed the −12 cut probe(same sequence as oligo 073-073-03).

[1068] For transcription, the dried samples were each dissolved in 4 μlof a solution containing 1 pmole copy template oligo 150 and 2 pmoles−11 partial promoter oligo 073-073-012 (SEQ ID NO: 131). Control samples3 and 4 each contained 1 pmole of the copy template oligo 150; sample 3also contained 1 pmole probe oligo 073-067-01 (SEQ ID NO: 132) and 2pmoles −11 partial promoter oligo 073-073-012 (see structure 88A);sample 4 contained 1 pmole −12 “cut” probe oligo 073-073-03 (SEQ ID NO:128) and 2 pmoles −11 partial promoter oligo 073-073-012 (see structure88B). These are the structures that would be expected to exist in thetranscription reactions from the two invasive cleavage reactionsdescribed above.

[1069] The transcription reactions were initiated, incubated, terminatedand the products were resolved and imaged as described in Ex. 40. Theresulting image is shown in the right half of FIG. 89 (lanes 6-9).Samples 3 and 4 appear in lanes 6 and 7, respectively, and the reactions1 and 2 from the invasive cleavage reaction products (indicated by theuse of the lower case “i”), appear in lanes 8 and 9, respectively. Thenumber of the Fig. showing the schematic representation of the expectedpromoter structure in each reaction is indicated above each lane, andthe placement of the nick is also indicated. The uppercase lettersindicate which structure in the particular Figure to examine for eachreaction. The lowercase “i” above lanes 8 and 9 indicate that thesetranscriptions were derived from actual invasive cleavage reactions.These products are compared to the RNA produced in the control reactionin lane 5, the procedure for which is described in Ex. 44. The RNAcreated by successful transcription appears in the upper third of thepanel (indicated by “RNA”).

[1070] The reaction shown in lane 6 shows no transcription. Thisdemonstrates that a nick between nucleotides −12 and −11 in theon-template strand of the T7 promoter eliminates transcription if thepromoter is assembled from uncut probe such as the 3′ end of the probeforms a branch within the promoter sequence. This is in contrast tot heresults seen with the −11/−10 nick examined below. Further, thetranscript apparent in lane 7 shows that an unbranched promoter with anick at the same site (−12/−11) produces the correct RNA, with fewabortive initiation products (see lanes 2 and 5 of FIG. 89, described inEx. 44). The reactions in lanes 8 and 9 demonstrate that the same effectis observed when the invasive cleavage reaction is the sole source ofthe upstream piece (−12 cut probe) of the T7 promoter. It is worthy ofnote that the promoter that is transcribed in lane 8 is made complete bythe presence of 1 pmole of a synthetic “cut” probe oligo, without anyuncut probe in the mixture, while the promoter that is transcribed inlane 9 is completed by the product of an invasive cleavage reaction thathad only 100 fmole of target DNA in it. This reaction also included theresidual uncut probe (up to approx. 10 pmoles), which may compete forbinding at the same site. Nonetheless, the transcriptions from theinvasive cleavage reaction products are only slightly reduced inefficiency, and are just as free of background as is the “no target”sample (lane 8). This Example clearly demonstrates that the cleavageproducts from the invasive cleavage reaction can be used in combinationwith a partial promoter oligo to promote the production of RNA, withoutbackground transcription generated by the presence of the uncut probe.This RNA product is clearly dependent on the presence of the targetmaterial in the invasive cleavage reaction.

Example 43

[1071] Shutting Down Transcription from A “Leaky” Branched T7 CompositePromoter Through the use of a Downstream Partial PromoterOligonucleotide having a 5′ Tail

[1072] The previous Example demonstrated that placement of a nick in thenon-template strand of a bacteriophage T7 promoter between the −12 and−11 nucleotides, relative to the transcription start site, preventstranscription of the branched promoter while allowing transcription whenthe composite promoter is assembled using the cut probe. When the nickis placed in other locations in the T7 promoter, transcription may beinitiated from either promoter, although it is usually less efficientfrom the branched promoter. This Example demonstrates that the additionof a 5′ tail that can base pair to the uncut probe (FIG. 90A) to thedownstream partial promoter piece effectively blocks transcription fromthat promoter, but does not prevent transcription when a cut probecompletes the promoter (FIG. 90B).

[1073] Two invasive cleavage reactions were set up, one without (rxn. 7)and one with (rxn. 8) input target DNA. The reactions (7 and 8)comprised 10 mM MOPS (pH 7.5), 0.05% Tween-20, 0.05% NP-40 and 20 pmolesprobe oligo 073-067-01 (SEQ ID NO: 132)and 10 pmoles INVADER oligo073-067-02 (SEQ ID NO: 133) in a volume of 14 μl. Reaction 8 alsoincluded 100 fmoles M13mp18 ssDNA. The samples were placed at 60° C. and6 μl of a solution containing 20 ng of Mja FEN-1 and 40 mM Mg₂Cl wereadded to each sample to start the reactions. The samples were incubatedat 60° C. for 30 minutes and then stopped by the addition of 3 μl of2.5M NaOAc, 83 mM Na₂EDTA (pH.8.0). Each sample was transferred to a 1.5ml microcentrifuge tube and the DNAs were precipitated, washed and driedas described in Ex. 42. The product of this invasive cleavage reactionis 13 nt oligonucleotide sequence, 5′-CGAAATTAATACG-3′ (SEQ ID NO: 127),termed the −11 cut probe (same sequence as oligo 073-061-01 which isreferred to as the −11 “cut” probe to indicate it was not generated inan invasive cleavage reaction).

[1074] In the transcription reactions, all of the DNAs were dissolved in4 μl of RNase-free dH₂O. Sample 1 had no added DNA, samples 2-8contained 1 pmole of the copy template oligo 150 (SEQ ID NO: 123). Inaddition, sample 3 contained 1 pmole of −11 “cut” probe oligo 073-061-01(SEQ ID NO: 127) and 2 pmoles of −10 partial promoter oligo 073-061-02(SEQ ID NO: 130), sample 4 contained 1 pmole of probe oligo 073-067-01and 2 pmoles of −10 partial promoter oligo 073-061-02. Control sample 5contained 1 pmole of probe oligo 073-067-01 and 2 pmoles of partialpromoter w/5′ tail oligo 073-074 (5′-TACTGACTCACTATAGGGTCTTCTATGGAGGTC-3′ (SEQ ID NO: 146) (see structure in FIG. 90A) and sample 6contained 1 pmole of −11 “cut” probe oligo 073-061-01 and 2 pmoles ofpartial promoter w/5′ tail oligo 073-074 (see structure in FIG. 90B).These are the structures (i.e., 90A and 90B) that would be expected toexist in the transcription reactions from the two invasive cleavagereactions described above.

[1075] The dried samples 7 and 8 from the invasive cleavage (above) wereeach dissolved in 4 μl of dH₂O containing 1 pmole copy template oligo150 and 2 pmoles partial promoter w/5′ tail oligo 073-074. Thetranscription reactions were initiated, incubated, terminated and thereaction products were resolved and imaged as described in Ex. 40. Theresulting image is shown in FIG. 91.

[1076] In FIG. 91 the lane numbers correspond to the sample numbers; thenumber of the Figure showing the schematic representation of theexpected promoter structure in each reaction is indicated above eachlane (“88” and “90”), and the placement of the nick is also indicated(“−11/−10”). The upper-case letters indicate which structure in theparticular Figure to examine for each reaction. The lower case “i” abovelanes 7 and 8 indicates that these transcriptions were derived fromactual invasive cleavage reactions. The RNA created by successfultranscription appears in the upper third of the panel, as indicated(“RNA”).

[1077] The control reactions in lanes 1 and 2, having either no DNA orhaving the only the copy template, produced no RNA as expected. Theproduct in lane 4 demonstrates that the branched T7 promoter with a nickin the non-template strand between nucleotides −11 and −10 can supporttranscription, albeit not as efficiently as the un-branched promoterwith the nick at the same site (lane 3). Examination of lane 5 showsthat the use of a partial promoter oligonucleotide with a short 5′ tailthat can basepair to the uncut probe as depicted in FIG. 90A,effectively suppresses this transcription but allows transcription whenthe probe does not have a 3′ tail (lane 6; schematic FIG. 90B). Thereactions in lanes 7 and 8 demonstrate that the same effect as observedwhen the invasive cleavage reaction is the sole source of the upstreampiece (−11 cut probe, SEQ ID NO: 127) of the T7 promoter. It is worthyof note that the promoter that is transcribed in sample 6 is madecomplete by the presence of 1 pmole of a synthetic “cut probe”, withoutany uncut probe in the mixture, while the promoter that is transcribedin sample 8 is completed by the product of an invasive cleavage reactionthat had only 100 fmole of target DNA in it. This reaction also includedthe residual uncut probe (up to approximately 19 pmoles), which maycompete for binding at the same site. Nonetheless, the transcriptionsfrom the invasive cleavage reaction products are just as strong and justas free of background in the “no target” samples.

[1078] This Example clearly demonstrates that the cleavage products fromthe invasive cleavage reaction can be used in combination with a partialpromoter oligonucleotide having a 5′ tail to promote the production ofRNA, without background transcription generated by the uncut probe. ThisRNA product is clearly dependent on the presence of the target materialin the invasive cleavage reaction.

Example 44

[1079] Creation of a Complete Bacteriophage T7 Promoter by DNAPolymerase-Mediated Extension of a Cut Probe Comprising a Partial T7Promoter

[1080] As demonstrated in the Examples above, transcription cannot occurfrom the T7 promoter unless a complete promoter region is present. Inthe above Examples, a complete promoter containing a nick in one strandwas created by annealing a cut probe generated from an invasive cleavagereaction to a copy template that was annealed to a partial promoteroligo. An alternative means of creating a complete promoter in a mannerdependent upon detection of a target sequence in an invasive cleavagereaction is to anneal the cut probe to a copy template devoid of apartial promoter oligo. The 3′-OH present at the end of the annealed cutprobe is then extended by a DNA polymerase to create a complete andun-nicked promoter that is transcription-competent.

[1081] In this Example, the promoter was made complete through the useof primer extension, rather that by the co-hybridization of anotheroligonucleotide. The reaction steps are diagrammed schematically in FIG.87. Two invasive cleavage reactions were set up, one without (rxn. 1)and one with (rxn. 2) input target DNA. The reactions (1 and 2)comprised 10 mM MOPS (pH 7.5), 0.05% Tween-20, 0.05% NP-40 and 20 pmolesprobe oligo 073-067-01 (SEQ ID NO: 132) and 10 pmoles INVADER oligo073-073-02 (SEQ ID NO: 134) in a volume of 14 μl. Reaction 2 alsoincluded 100 fmoles M13mp18 ssDNA. The samples were placed at 60° C. and6 μl of a solution containing 20 ng of Mja FEN-1 and 40 mM Mg₂Cl wereadded to each sample to start the reactions. The samples were incubatedat 60° C. for 30 minutes and stopped by the addition of 3 μl of 2.5MNaOAc, 83 mM Na₂EDTA (pH 8.0). Each sample was transferred to a 1.5 mlmicrocentrifuge tube and then the DNAs were precipitated, washed anddried as described in Ex. 42. The product of this invasive cleavagereaction is the 12 nt oligonucleotide sequence: 5′-CGAAATTAATAC-3′ (SEQID NO: 128), termed the −12 cut probe (same sequence as oligo 073-073-03which is referred to as the −12 “cut” probe to indicate it was notgenerated in an invasive cleavage reaction).

[1082] To allow extension of these products using a template-dependentDNA polymerase, a 20 μl solution containing 20 mM Tris-HCl (pH 8.5), 1.5mM Mg₂Cl, 50 mM KCl, 0.05% Tween-20, 0.05% NP-40, 25 μM each dNTP, 0.25units Taq DNA polymerase (Boehringer) and 2 μM copy template oligo 150(SEQ ID NO: 123) was added to each of the dried cleavage samples. Thesamples were incubated at 30° C. for 1 hr. The primer extensionreactions were stopped by the addition of 3 μl of 2.5M NaOAc with 83 mMNa₂EDTA (pH 8.0)/sample. Each sample was transferred to a 1.5 mlmicrocentrifuge tube and the DNAs were precipitated, washed and dried asdescribed in Ex. 42.

[1083] Samples 1 and 2 were then dissolved in 4 μl RNase-free dH₂O.Samples 3, 4 and 5 are control reactions: sample 3 was 4 μl ofRNase-free dH₂O without added DNA, sample 4 contained 1 pmole of thecopy template oligo 150 (SEQ ID NO: 123) in 4 μl of RNase-free dH₂O, andsample 5 contained 1 pmole of the same copy template and 1 pmole of thecomplete promoter oligo 151 (SEQ ID NO: 124) in RNase-free dH₂O.

[1084] Transcription reactions were performed using the MEGAshortscript™system, in accordance with the manufacturer's instructions, but with theaddition of a fluorescein labeled ribonucleotide. To each sample, 6 μlof a solution containing 1 μl of 10× Transcription Buffer, 7.5 mM eachrNTP, 0.125 mM fluorescein-12-UTP (Boehringer) and 1 μl T7MEGAshortscript™ Enzyme Mix was added. The samples were incubated at 37°C. for 1 hour. One μl of RNase-free DNase 1 (2U/μl) was added to eachsample and they were incubated an additional 15 minutes at 37° C. Thereactions were stopped by the addition of 10 μl of a solution of 95%formamide, 5 mM NaEDTA, with loading dyes. All samples were heated to95° C. for 2 minutes and four μl of each sample were resolved byelectrophoresis through a 20% denaturing acrylamide gel (19:1cross-linked) with 7 M urea, in a buffer containing 45 mM Tris-Borate(pH 8.3), 1.4 mM EDTA. The results were imaged using the MolecularDynamics Fluoroimager 595, with excitation at 488 nm and, emissiondetected at 530 nm.

[1085] The resulting image is shown in lanes 1 through 5 of FIG. 89; thelane numbers correspond to the sample numbers. The Figure numberscorresponding to the schematic representations of the promoterstranscribed in each reaction as indicated above the lanes. The RNAproduct from successful transcription appears in the upper third of thepanel, as indicated (“RNA”). Unincorporated labeled nucleotide appearsas a dense signal near the bottom (“NTPs”). Short transcription productscaused by aborted initiation events (Milligan and Uhlenbeck, MethodsEnzymol., 180:51 [1989]) appear as bands just above the free nucleotidein the lanes showing active transcription (i.e., lanes 2 and 5).

[1086] It can clearly be seen from the data in lanes 1 and 2 that thetranscription is dependent on the presence of the target material in theinvasive cleavage reaction. It is shown elsewhere (see lane 3, FIG. 92)that the product of the cleavage reaction is not in itself sufficient toallow transcription from the copy template. Thus, the action of the DNApolymerase in extending the hybridized cut probe across the promoter isa necessary step in enabling the transcription in this embodiment. Thesedata clearly demonstrate that both template-dependent extension by DNApolymerase, and extension followed by transcription are suitable methodsof visualizing the products of the invasive cleavage assay. As discussedin the Description of the Invention, the products of thermal breakdownthat possess 3′ terminal phosphates would not be extended, and wouldthus be precluded from contributing to background transcription.

Example 45

[1087] Test for the Dependence of an Enzyme on the Presence of anUpstream Oligonucleotide

[1088] When choosing a structure-specific nuclease for use in asequential invasive cleavage reaction it is preferable that the enzymehave little ability to cleave a probe 1) in the absence of an upstreamoligonucleotide, and 2) in the absence of overlap between the upstreamoligonucleotide and the downstream labeled probe oligonucleotide. FIGS.99a-e depicts the several structures that can be used to examine theactivity of an enzyme that is confronted with each of these types ofstructures. The structure a (FIG. 99a) shows the alignment of a probeoligonucleotide with a target site on bacteriophage M13 DNA (M13sequences shown in FIG. 99 are provided in SEQ ID NO: 163) in theabsence of an upstream oligonucleotide. Structure b (FIG. 99b) isprovided with an upstream oligonucleotide that does not contain a regionof overlap with the labeled probe (the label is indicated by the star).In structures c, d and e (FIGS. 99c-e) the upstream oligonucleotideshave overlaps of 1, 3 or 5 nucleotides, respectively, with thedownstream probe oligonucleotide and each of these structures representsa suitable invasive cleavage structure. The enzyme Pfu FEN-1 was testedfor activity on each of these structures and all reactions wereperformed in duplicate.

[1089] Each reaction comprised 1 μM 5′ TET labeled probe oligonucleotide89-15-1 (SEQ ID NO: 152), 50 nM upstream oligonucleotide (either oligo81-69-2 [SEQ ID NO: 153], oligo 81-69-3 [SEQ ID NO: 154], oligo 81-69-4[SEQ ID NO: 155], oligo 81-69-5 [SEQ ID NO: 156], or no upstreamoligonucleotide), 1 fmol M13 target DNA, 10 mg/ml tRNA and 10 ng of PfuFEN-1 in 10 μl of 10 mM MOPS (pH 7.5), 7.5 mM MgCl₂ with 0.05% each ofTween 20 and Nonidet P-40.

[1090] All of the components except the enzyme and the MgCl₂ wereassembled in a final volume of 8 μl and were overlaid with 10 μl ofChill-Out™ liquid wax. The samples were heated to the reactiontemperature of 69° C. The reactions were started by the addition of thePfu FEN-1 and MgCl₂, in a 2 μl volume. After incubation at 69° C. for 30minutes, the reactions were stopped with 10 μl of 95% formamide, 10 mMEDTA, 0.02% methyl violet. Samples were heated to 90° C. for 1 minimmediately before electrophoresis through a 20% denaturing acrylamidegel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. Gels were then analyzed with aFMBIO-100 Hitachi FMBIO fluorescence imager. The resulting image isdisplayed in FIG. 100.

[1091] In FIG. 100, lanes labeled “a” contain the products generatedfrom reactions conducted without an upstream oligonucleotide (structurea), lanes labeled “b” contain an upstream oligonucleotide that does notinvade the probe/target duplex (structure b). Lanes labeled “c”, “d” and“e” contain the products generated from reactions conducted using anupstream oligonucleotide that invades the probe/target duplex by 1, 3 or5 bases, respectively. The size (in nucleotides) of the uncleaved probeand the cleavage products is indicated to the left of the image in FIG.100.

[1092] As shown in FIG. 100, cleavage of the probe was not detectablewhen structures a and b were utilized. In contrast, cleavage productswere generated when invasive cleavage structures were utilized(structures c-e). These data show that the Pfu FEN-1 enzyme requires anoverlapping upstream oligonucleotide for specific cleavage of the probe.

[1093] Any enzyme may be examined for its suitability for use in asequential invasive cleavage reaction by examining the ability of thetest enzyme to cleave structures a-e (it is understood by those in theart that the specific oligonucleotide sequences shown in FIGS. 99a-eneed not be employed in the test reactions; these structures are merelyillustrative of suitable test structures). Desirable enzymes displaylittle or no cleavage of structures a and b and display specificcleavage of structures c-e (i.e., they generate cleavage products of thesize expected from the degree of overlap between the twooligonucleotides employed to form the invasive cleavage structure).

Example 46

[1094] Use of the Products of a First Invasive Cleavage Reaction toEnable a Second Invasive Cleavage Reaction with a Net Gain inSensitivity

[1095] As discussed in the Description of The Invention above, thedetection sensitivity of the invasive cleavage reaction can be increasedby the performing a second round of invasive cleavage using the productsof the first reaction to complete the cleavage structure in the secondreaction (shown schematically in FIG. 96). In this Example, the use of aprobe that, when cleaved in a first invasive cleavage reaction, forms anintegrated INVADER oligo and target molecule for use in a secondinvasive cleavage reaction, is illustrated (shown schematically in FIG.97).

[1096] A first probe was designed to contain some internalcomplementarity so that when cleaved in a first invasive cleavagereaction the product (“Cut Probe 1”) could form a target strandcomprising an integral INVADER oligonucleotide, as depicted in FIG. 97.A second probe was provided in the reaction that would be cleaved at theintended site when hybridized to the newly formed target/INVADER (FIG.97). To demonstrate the gain in signal due to the performance ofsequential invasive cleavages, a standard invasive cleavage assay, asdescribed above, was performed in parallel.

[1097] All reactions were performed in duplicate. Each standard (i.e.,non-sequential) invasive cleavage reaction comprised 1 μM 5′fluorescein-labeled probe oligo 073-182 (5′Fl-AGAAAGGAAGGGAAGAAAGCGAA-3′; SEQ ID NO: 157), 10 nM upstream oligo81-69-4 (5′-CTTGACGGGGAAAGCCGGCGAACGTGGCGA-3′; SEQ ID NO: 155), 10 to100 attomoles of M13 target DNA, 10 mg/ml tRNA and 10 ng of Pfu FEN-1 in10 μl of 10 mM MOPS (pH 7.5), 8 mM MgCl₂ with 0.05% each of Tween 20 andNonidet P-40. All of the components except the enzyme and the MgCl₂ wereassembled in a volume of 7 μl and were overlaid with 10 μl of Chill-Out™liquid wax. The samples were heated to the reaction temperature of 62°C. The reactions were started by the addition of the Pfu FEN-1 andMgCl₂, in a 2 μl volume. After incubation at 62° C. for 30 minutes, thereactions were stopped with 10 μl of 95% formamide, 10 mM EDTA, 0.02%methyl violet.

[1098] Each sequential invasive cleavage reaction comprised 1 μM 5′fluorescein-labeled oligonucleotide 073-191 (the first probe or “Probe1”, 5′ Fl-TGGAGGTCAAAACATCG ATAAGTCGAAGAAAGGAAGGGAAGAAAT-3′; SEQ ID NO:158), 10 nM upstream oligonucleotide 81-69-4 (5′-CTTGACGGGGAAAGCCGGCGAACGTGGCGA-3′; SEQ ID NO: 155), 1 μM of 5′ fluorescein labeledoligonucleotide 106-32 (the second probe or “Probe 2”, 5′ Fl-TGTTTTGACCTCCA-3′; SEQ ID NO: 159), 1 to 100 amol of M13 target DNA, 10 mg/ml tRNAand 10 ng of Pfu FEN-1 in 10 μl of 10 mM MOPS (pH 7.5), 8 mM MgCl₂ with0.05% each of Tween 20 and Nonidet P-40. All of the components exceptthe enzyme and the MgCl₂ were assembled in a volume of 8 μl and wereoverlaid with 10 μl of Chill-Out™ liquid wax. The samples were heated tothe reaction temperature of 62° C. (this temperature is the optimumtemperature for annealing of Probe 1 to the first target). The reactionswere started by the addition of Pfu FEN-1 and MgCl₂, in a 2 μl volume.After incubation at 62° C. for 15 minutes, the temperature was loweredto 58° C. (this temperature is the optimum temperature for annealing ofProbe 2 to the second target) and the samples were incubated for another15 min. Reactions were stopped by the addition of 10 μl of 95%formamide, 20 mM EDTA, 0.02% methyl violet.

[1099] Samples from both the standard and the sequential invasivecleavage reactions were heated to 90° C. for 1 min immediately beforeelectrophoresis through a 20% denaturing acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA. The gel was then analyzed with a Molecular DynamicsFluorImager 595. The resulting image is displayed in FIG. 101a. A graphshowing measure of fluorescence intensity for each of the product bandsis shown in FIG. 101b.

[1100] In FIG. 101a, lanes 1-5 contain the products generated instandard invasive cleavage reactions that contained either no target(lane 1), 10 amol of target (lanes 2 and 3) or with 100 amol of target(lanes 4 and 5). The uncleaved probe is seen as a dark band in each laneabout half way down the panel and the cleavage products appear as asmaller black band near the bottom of the panel, the position of thecleavage product is indicated by an arrow head to the left of FIG. 101a.The gray ladder of bands seen in lanes 1-5 is due to the thermaldegradation of the probe as discussed above and is not related to thepresence or absence of the target DNA. The remaining lanes displayproducts generated in sequential invasive cleavage reactions thatcontained 1 amol of target (lanes 6 and 7), 10 amol of target (lanes 8and 9) and 100 amol of target (lanes 10 and 11). The uncleaved firstprobe (Probe 1; labeled “1 uncut”) is seen near the top of the panel,while the cleaved first probe is indicated as “1: cut”. Similarly, theuncleaved and cleaved second probe are indicated as “2: uncut” and 2:cut,” respectively.

[1101] The graph shown in FIG. 101b compares the amount of productgenerated from the standard reaction (“Series 1”) to the amount ofproduct generated from the second step of the sequential reaction(“Series 2”). The level of background fluorescence measured from areaction that lacked target DNA was subtracted from each measurement. Itcan be seen from the table located below the graph that the signal fromthe standard invasive cleavage assay that contained 100 attomoles oftarget DNA was nearly identical to the signal from the sequentialinvasive cleavage assay in which 1 attomole of target was present,indicating that the inclusion of a second cleavage structure increasesthe sensitivity of the assay 100 to 200-fold. This boost in signalallows easy detection of target nucleic acids at the sub attomole levelusing the sequential invasive cleavage assay, while the standard assay,when performed using this enzyme for only 30 minutes, does not generatedetectable product in the presence of 10 attomoles of target.

[1102] When the amount of target was decreased by 10 or 100 fold in thesequential invasive cleavage assay, the intensity of the signal wasdecreased by the same proportion. This indicates that the quantitativecapability of the invasive cleavage assay is retained even whenreactions are performed in series, thus providing a nucleic aciddetection method that is both sensitive and quantitative.

[1103] While in this Example, the two probes used had different optimalhybridization temperatures (i.e., the temperature empiracally determinedto give the greatest turnover rate in the given reaction conditions),the probes may also be selected (i.e., designed) to have the sameoptimal hybridization temperature so that a temperature shift duringincubation is not necessary.

Example 47

[1104] The Products of a Completed Sequential Invasive Cleavage ReactionCannot Cross Contaminate Subsequent Similar Reactions

[1105] As discussed in the Description of the Invention, the serialnature of the multiple invasive cleavage events that occur in thesequential invasive cleavage reaction, in contrast to the reciprocatingnature of the polymerase chain reaction and similar doubling assays,means that the sequential invasive cleavage reaction is not subject tocontamination by the products of like reactions because the products ofthe first cleavage reaction do not participate in the generation of newsignal in the second cleavage reaction. If a large amount of a completedreaction were to be added to a newly assembled reaction, the backgroundthat would be produced would come from the amount of target that wasalso carried in, combined with the amount of already-cleaved probe thatwas carried in. In this Example, it is demonstrated that a very largeportion of a primary reaction must be introduced into the secondaryreaction to create significant signal.

[1106] A first or primary sequential invasive cleavage reaction wasperformed as described above using 100 amol of target DNA. A second setof 5 reactions were assembled as described in Ex. 46 with the exceptionthat portions of the first reaction were introduced and no additionaltarget DNA was included. These secondary reactions were initiated andincubated as described above, and included 0, 0.01, 0.1, 1, or 10% ofthe first reaction material. A control reaction including 100 amol oftarget was included in the second set also. The reactions were stopped,resolved by electrophoresis and visualized as described above, and theresulting image is displayed in FIG. 102. The primary probe, uncutsecond probe and the cut 2nd probe are indicated on the left as “1:cut”, 2: uncut” and 2: cut”, respectively.

[1107] In FIG. 102, lane 1 shows the results of the first reaction withthe accumulated product at the bottom of the panel, and lane 2 show a1:10 dilution of the same reaction, to demonstrate the level of signalthat could be expected from that level of contamination, without furtheramplification. Lanes 3 through 7 show the results of the secondarycleavage reactions that contained 0, 10, 1, 0.1 or 0.01% of the firstreaction material added as contaminant, respectively and lane 8 shows acontrol reaction that had 100 amol of target DNA added to verify theactivity of the system in the secondary reaction. The signal level inlane 4 is as would be expected when 10% of the pre-cleaved material istransferred (as in lane 2) and 10% of the transferred target materialfrom the lane 1 reaction is allowed to further amplify. At all levels offurther dilution the signal is not readily distinguished frombackground. These data demonstrate that while a large-scale transferfrom one reaction to another may be detectable, cross contamination bythe minute quantities that would be expected from aerosol or fromequipment contamination would not be easily mistaken for a falsepositive result. These data also demonstrate that when the products ofone reaction are deliberately carried over into a fresh sample, theseproducts do not participate in the new reaction, and thus do not affectthe level of target-dependent signal that may be generated in thatreaction.

Example 48

[1108] Detection of Human Cytomegalovirus Viral DNA by Invasive Cleavage

[1109] The previous Example demonstrates the ability of the invasivecleavage reaction to detect minute quantities of viral DNA in thepresence of human genomic DNA. In this Example, the probe and INVADERoligonucleotides were designed to target the 3104-3061 region of themajor immediate early gene of human cytomegalovirus (HCMV) as shown inFIG. 103. In FIG. 103, the INVADER oligo (89-44; SEQ ID NO: 160) and thefluorescein (Fl)-labeled probe oligo (89-76; SEQ ID NO: 161) are shownannealed along a region of the HCMV genome corresponding to nucleotides3057-3110 of the viral DNA (SEQ ID NO: 162). The probe used in thisExample is a poly-pyrimidine probe and as shown herein the use of apoly-pyrimidine probe reduces background signal generated by the thermalbreakage of probe oligos.

[1110] The genomic viral DNA was purchased from AdvancedBiotechnologies, Incorporated (Columbia, Md.). The DNA was estimated(but not certified) by personnel at Advanced Biotechnologies to be at aconcentration of 170 amol (1×10⁸ copies) per microliter. The reactionswere performed in quadruplicate. Each reaction comprised 1 μM 5′fluorescein labeled probe oligonucleotide 89-76 (SEQ ID NO: 161), 100 nMINVADER oligonucleotide 89-44 (SEQ ID NO: 160), 1 ng/ml human genomicDNA, and one of five concentrations of target HCMV DNA in the amountsindicated above each lane in FIG. 104, and 10 ng of Pfu FEN-1 in 10 μlof 10 mM MOPS (pH 7.5), 6 mM MgCl₂ with 0.05% each of Tween 20 andNonidet P-40. All of the components except the labeled probe, enzyme andMgCl₂ were assembled in a final volume of 7 μl and were overlaid with 10μl of Chill-Out™ liquid wax. The samples were heated to 95° C. for 5min, then reduced to 62° C. The reactions were started by the additionof probe, Pfu FEN-1 and MgCl₂, in a 3 μl volume. After incubation at 62°C. for 60 minutes, the reactions were stopped with 10 μl of 95%fonnamide, 10 mM EDTA, 0.02% methyl violet. Samples were heated to 90°C. for 1 min immediately before electrophoresis through a 20% acrylamidegel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. Gels were then analyzed with aMolecular Dynamics FluorImager 595.

[1111] The resulting image is displayed in FIG. 104. The replicatereactions were run in groups of four lanes with the target HCMV DNAcontent of the reactions indicated above each set of lanes (0-170 amol).The uncleaved probe is seen in the upper third of the panel (“Uncut89-76”) while the cleavage products are seen in the lower two-thirds ofthe panel (“Cut 89-76”). It can be seen that the intensity of theaccumulated cleavage product is proportional to the amount of the targetDNA in the reaction. Furthermore, it can be clearly seen in reactionsthat did not contain target DNA (“no target”) that the probe is notcleaved, even in a background of human genomic DNA. While 10 ng of humangenomic DNA was included in each of the reactions shown in FIG. 104,inclusion of genomic DNA up to 200 ng has slight impact on the amount ofproduct accumulated. The data did not suggest that 200 ng per 10 μl ofreaction mixture represented the maximum amount of genomic DNA thatcould be tolerated without a significant reduction in signalaccumulation. For reference, this amount of DNA exceeds what might befound in 0.2 ml of urine (a commonly tested amount for HCMV in neonates)and is equivalent to the amount that would be found in about 5 μl ofwhole blood.

[1112] These results demonstrate that the standard (i.e.,non-sequential) invasive cleavage reaction is a sensitive, specific andreproducible means of detecting viral DNA. It can also be seen fromthese data that the use of a poly-pyrimidine probe reduces thebackground from thermal breakage of the probe, as discussed in Example22. Detection of 1.7 amol of target is roughly equivalent to detectionof 10⁶ copies of the virus. This is equivalent to the number of viralgenomes that might be found in 0.2 mls of urine from a congenitallyinfected neonate (102 to 106 genome equivalents per 0.2 mls; Stagno etal., J. Infect. Dis., 132:568 [1975]). Use of the sequential invasivecleavage assay would permit detection of even fewer viral DNA molecules,facilitating detection in blood (10¹ to 10⁵ viral particles per ml;Pector et al., J. Clin. Microbiol., 30:2359 [1992]), which carries amuch larger amount of heterologous DNA.

[1113] From the above it is clear that the invention provides reagentsand methods to permit the detection and characterization of nucleic acidsequences and variations in nucleic acid sequences. The INVADER-directedcleavage reaction and the sequential INVADER-directed cleavage reactionof the present invention provide ideal direct detection methods thatcombine the advantages of the direct detection assays (e.g., easyquantification and minimal risk of carry-over contamination) with thespecificity provided by a dual or tri oligonucleotide hybridizationassay.

[1114] As indicated in the Description of the Invention, the use ofsequential invasive cleavage reactions can present the problem ofresidual uncut first, or primary, probe interacting with the secondarytarget, and either competing with the cut probe for binding, or creatingbackground through low level cleavage of the resulting structure. Thisis shown diagramatically in FIGS. 105 and 106. In FIG. 105, the reactiondepicted makes use of the cleavage product from the first cleavagestructure to form an INVADER oligonucleotide for a second cleavagereaction. The structure formed between the secondary target, thesecondary probe and the uncut primary probe is depicted in FIG. 105, asthe right hand structure shown in step 2a. This structure is recognizedand cleaved by the 5′ nucleases, albeit very inefficiently (i.e., atless than about 1% in most reaction conditions). Nonetheless, theresulting product is indistinguishable from the specific product, andthus may lead to a false positive result. The same effect can occur whenthe cleaved primary probe creates and integrated INVADER/target (IT)molecule, as described in Example 46; the formation of the undesirablecomplex is depicted schematically in FIG. 106, as the right handstructure shown in step 2a.

[1115] The improvements provided by the inclusion of ARRESTORoligonucleotides of various compositions in each of these types ofsequential INVADER assays are demonstrated in the following Examples.These ARRESTOR oligonucleotides are configured to bind the residualuncut probe from the first cleavage reaction in the series, therebyincreasing the efficacy of and reducing the non-specific background inthe subsequent reaction(s).

Example 49

[1116] “ARRESTOR” Oligonucleotides Improve Sensitivity of MultipleSequential Invasive Cleavage Assays

[1117] In this Example, the effect of including an ARRESTORoligonucleotide on the generation of signal using the IT probe systemdepicted in FIGS. 97 and 106 is demonstrated. The ARRESTORoligonucleotide hybridizes to the primary probe, mainly in the portionthat recognizes the target nucleic acid during the first cleavagereaction. In addition to examining the effects of adding an ARRESTORoligonucleotide, the effects of using ARRESTOR oligonucleotides thatextended in complementarity different distances into the region of theprimary probe that composes the secondary IT structure were alsoinvestigated. These effects were compared in reactions that included thetarget DNA over a range of concentrations, or that lacked target DNA, inorder to demonstrate the level of nonspecific (i.e., not related totarget nucleic acid) background in each set of reaction conditions.

[1118] The target DNA for these reactions was a fragment that comprisedthe full length of the hepatitis B genome from strain of serotype adw.This material was created using the polymerase chain reaction fromplasmid pAM6 (ATCC #45020D). The PCRs were conducted using avector-based forward primer, oligo #156-022-001(5′-ggcgaccacacccgtcctgt-3′; SEQ ID NO: 168) and a reverse primer, oligo#156-022-02 (5′-ccacgatgcgtccggcgtag-3′; SEQ ID NO: 169) to amplify thefull length of the HBV insert, an amplicon of about 3.2 kb. The cyclingconditions included a denaturation of the plasmid at 95° C. for 5minutes, followed by 30 cycles of 95° C., 30 seconds; 60° C., 40seconds; and 72° C., 4 minutes. This was followed by a final extensionat 72° C. for 10 minutes. The resulting amplicon, termed pAM6#2, wasadjusted to 2 M NH₄OAc, and collected by precipitation wiht isopropanol.After drying in vacuo, DNA was dissolved in 10 mM Tris pH.0, 0.1 mMEDTA. The concentration was determined by OD₂₀₀ measurement, and byINVADER assay with comparison to a standard of known concentration.

[1119] The INVADER reactions were conducted as follows. Five mastermixes, termed “A,” “B,” “C,” “D,” and “E,” were assembled; all mixescontained 12.5 mM MOPS, pH 7.5, 500 fmoles primary INVADER oligo#218-55-05 (SEQ ID NO: 171), 10 ng human genomic DNA (Novagen) and 30 ngAfuFEN1 enzyme, for every 8 μl of mix. Mix A contained no added HBVgenomic amplicon DNA; mix B contained 600 molecules of HBV genomicamplicon DNA pAM6 #2; mix C contained 6,000 molecules pAM6 #2; mix Dcontained 60,000 molecules pAM6 #2; and mix E contained 600,000molecules pAM6 #2. The mixes were aliquotted to the reaction tubes, 8μl/tube: mix A to tubes 1, 2, 11, 12, 21 and 22; mix B to tubes 3, 4,13, 14, 23 and 24; mix C to tubes 5, 6, 15, 16, 25 and 26; mix D totubes 7, 8, 17, 18, 27 and 28; and mix E to tubes 9, 10, 19, 20, 29 and30. The samples were incubated at 95° C. for 4 minutes to denature theHBV genomic amplicon DNA. The reactions were then cooled to 67° C., and2 μl of a mix containing 37.5 mM MgCl₂ and 2.5 pmoles 218-95-06 (SEQ IDNO: 183) for every 2 μl, was added to each sample. The samples wereincubated at 67° C. for 60 minutes. Three secondary reaction mastermixes were prepared, all mixes contained 10 pmoles of secondary probeoligonucleotide #228-48-04 (SEQ ID NO: 173) for every 2 μl of mix. Mix2A contained no additional oligonucleotide, mix 2B contained 5 pmoles“ARRESTOR” oligo #218-95-03 (SEQ ID NO: 184) and mix 2C contained 5pmoles of“ARRESTOR” oligo #218-95-01 (SEQ ID NO: 174). After the 60minute incubation at 67° C. (the primary reaction described above), 2 μlof the secondary reaction mix was added to each sample: Mix 2A was addedto samples #1-10; Mix 2B was added to samples #11-20; and Mix 2C wasadded to samples #21-30. The temperature was adjusted to 52° C. and thesamples were incubated for 30 minutes at 52° C. The reactions were thenstopped by the addition of 10 μl of a solution of 95% formamide, 5 mMEDTA and 0.02% crystal violet. All samples were heated to 95° C. for 2minutes, and 4 μl of each sample were resolved by electrophoresisthrough 20% denaturing acrylamide gel (19:1 cross-linked) with 7 M urea,in a buffer containing 45 mM Tris-Borate (pH8.3) and 1.4 mM EDTA. Theresults were imaged using the Molecular Dynamics Fluoroimager 595,excitation 488, emission 530. The resulting images are shown in FIG.107.

[1120] In FIG. 107, Panel A shows the results of the target titrationwhen no ARRESTOR oligonucleotide was included in the secondary reaction;Panel B shows the results of the same target titration using an ARRESTORoligonucleotide that extended 2 nt into the non-target complementaryregion of the primary probe; and Panel C shows the results of the sametarget titration using an ARRESTOR oligonucleotide that extended 4 ntinto the non-target complementary region of the primary probe. Theproduct of the secondary cleavage reaction is seen as a band near thebottom of each panel. The first two lanes of each panel (i.e., 1 and 2,11 and 12, 21 and 22) lacked target DNA, and the signal the co-migrateswith the product band represents the nonspecific background under eachset of conditions.

[1121] It can be seen by visual inspection of these panels that thebackground signal is both reduced, and made more predictable, by theinclusion of either species of ARRESTOR oligonucleotides. In addition toreducing the background in the no-target control lanes, the backgroundreduction in the reactions that had the more dilute amounts of targetincluded is reduced, leading to a signal that is a more accuratereflection of the target contained within the reaction, thus improvingthe quantitative range of the multiple, sequential invasive cleavagereaction.

[1122] To quantify the impact of including the ARRESTOR oligonucleotidein the secondary cleavage reaction under these conditions, the averageproduct band signal from the reactions having the largest amount oftarget (i.e., averages of the signals from lanes 9 and 10, lanes 19 and20, and lanes 29 and 30), were compared to the averaged signal from theno-target contol lanes for each panel, determine the “fold overbackground,” the factor of signal amplification over background, undereach set of conditions. For the reactions without the ARRESTORoligonucleotide, Panel A, the fold over background was 5.3; for Panel B,the fold over background was 12.7; and for Panel C., the fold overbackground was 13.4, indicating that in this system inclusion of anyARRESTOR oligonucleotide at least doubled the specificity of the signalover the ARRESTOR oligonucleotide -less reactions, and that the ARRESTORoligonucleotide that extended slightly farther into the non-targetcomplementary region may be slightly more effective, at least in thisembodiment of the system. This clearly shows the benefits of using anARRESTOR oligonucleotide to enhance the specificity of these reactions,an advantage that is of particular benefit at low levels of targetnucleic acid.

Example 50

[1123] “ARRESTOR” Oligonucleotides Allow Use of Higher Concentrations ofPrimary Probe Without Increasing Background Signal

[1124] It was demonstrated in Example 36, that increasing theconcentration of the probe in the invasive cleavage reaction coulddramatically increase the amount of signal generated for a given amountof target DNA. While not intending to limit the explanation to anyspecific mechanism, this is believed to be caused by the fact thatincreased concentration of probe increases the rate at which the cleavedprobe is supplanted by an uncleaved copy, thereby increasing theapparent turnover rate of the cleavage reaction. Unfortunately, thiseffect could not heretofore be applied in the primary cleavage reactionof a multiple sequential INVADER assay because the residual uncleavedprimary probe can hybridize to the secondary target, in competition withthe cleaved molecules, thereby reducing the efficacy of the secondaryreaction. Elevated concentrations of primary probe exacerbate thisproblem. Further, the resulting complexes, as described above, can becleaved at a low level, contributing to background. Therefore,increasing the primary probe can have the double negative effect of bothslowing the secondary reaction and increasing the level of this form ofnon target-specific background. The use of an ARRESTOR oligonucleotideto sequester or neutralize the residual primary probe allows thisconcentration-enhancing effect to be applied to these sequentialreactions.

[1125] To demonstrate this effect, two sets of reactions were conducted.In the first set of reactions, the reactions were conducted using arange of primary probe concentrations, but no ARRESTOR oligonucleotidewas supplied in the secondary reaction. In the second set of reactions,the same probe concentrations were used, but an ARRESTOR oligonucleotidewas added for the secondary reactions.

[1126] All reactions were performed in duplicate. Primary INVADERreactions were done in a final volume of 10 μl and contained: 10 mMMOPS, pH 7.5, 7.5 mM MgCl₂, 500 fm of primary INVADER (218-55-05; SEQ IDNO: 171); 30 ng of AfuFEN1 enzyme and 10 ng of human genomic DNA. 100zeptomoles of HBV pAM6 #2 amplicon was included in all even numberedreactions (by reference to FIGS. 108A and B). Reactions included 10pmoles, 20 pmoles, 50 pmoles, 100 pmoles or 150 pmoles of primary probe(218-55-02; SEQ ID NO: 170). MOPS, target and INVADER oligonucleotideswere combined to a final volume of 7 μl. Samples were heat denatured at95° C. for 5 minutes, then cooled to 67° C. During the 5 minutedenaturation, MgCl₂, probe and enzyme were combined. The primary INVADERreactions were initiated by the addition of 3 μl of MgCl₂, probe andenzyme mix, to the final concentrations indicated above. Reactions wereincubated for 30 minutes at 67° C. The reactions were then cooled to 52°C., and each primary INVADER reaction received the following secondaryreaction components in a total volume of 4 μl: 2.5 pmoles secondarytarget (oligo number 218-95-04; SEQ ID NO: 172); 10 pmoles secondaryprobe (oligo number 228-48-04; SEQ ID NO: 173). The reactions thatincluded the ARRESTOR oligonucleotide had either 40 pmoles, 80 pmoles,200 pmoles, 400 pmoles or 600 pmoles of ARRESTOR oligonucleotide (oligonumber 218-95-01; SEQ ID NO: 174), added at a 4-fold molar excess overthe primary probe amount for each reaction, with this mix. Reactionswere then incubated at 52° C. for 30 minutes. The reactions were stoppedby the addition of 10 μl of a solution of 95% formamide, 10 mM EDTA and0.02% crystal violet. All samples were heated to 95° C. for 1 minute,and 4 μl of each sample were resolved by electrophoresis through 20%denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffercontaining 45 mM Tris-Borate (pH8.3) and 1.4 mM EDTA. The results wereimaged using the Molecular Dynamics Fluoroimager 595, excitation 488,emission 530. The resulting images for the reactions either without orwith an ARRESTOR oligonucleotide are shown in FIGS. 108A and 108B,respectively. The products of cleavage of the secondary probe are seenas a band near the bottom of each panel.

[1127] In FIG. 108A, lane sets 1 and 2 show results with 10 pmoles ofprimary probe; 3 and 4 had 20 pmoles; 5 and 6 had 50 pmoles; 7 and 8 had100 pmoles; and 9 and 10 had 150 pmoles. It can be seen by visualexamination, that the increases in the amount of primary probe have thecombined effect of slightly increasing the background in the no-targetlanes (odd numbers) while reducing the specific signal in the presenceof target (even numbered lanes), and therefore the reducing thespecificity of the reaction if viewed as the measure of “fold overbackground,” demonstrating that the approach of increasing signal byincreasing probe cannot be applied in these sequential reactions.

[1128] In FIG. 108B, lane sets 1 and 2 show results with 10 pmoles ofprimary probe; while 3 and 4 had 20 pmoles; 5 and 6 had 50 pmoles; 7 and8 had 100 pmoles; and 9 and 10 had 150 pmoles. In addition, eachreaction included 4-fold molar excess of the ARRESTOR oligonucleotideadded before the secondary cleavage reaction. It can be seen by visualexamination that the background in the no-target lanes (odd numbers) islower in all cases, while the specific signal in the presence of target(even numbered lanes) increases with increased amounts of primary probe,leading to a greater “fold over background” sensitivity at this targetlevel.

[1129] To quantitatively compare these effects, the fluorescence signalfrom the products of both non-specific and specific cleavage weremeasured. The results are depicted graphically in FIG. 108C, graphed asa measure of the percentage of the secondary probe cleaved during thereaction, compared to the amount of primary probe used. Examination ofthe plots from the no-target reactions confirms that the background inthe absence of the ARRESTOR oligonucleotide is, in general, roughlytwo-fold higher, and that both increase slightly with the increasingprobe amounts. The specific signals however, diverge between the twosets of reaction more dramatically. While the signal in the no-ARRESTORoligonucleotide reactions decreases steadily as primary probe wasincreased, the signal in the ARRESTOR oligonucleotide reactionscontinued to increase. At the highest primary probe concentrationstested, the no-ARRESTOR oligonucleotide reactions had specific signalthat was only 1.7 fold over background, while the ARRESTORoligonucleotide reactions detected the 100 zmoles (60,000 copies) oftarget with a signal 6.5 fold over background, thus demonstrating theimprovement in the sequential invasive cleavage reaction when anARRESTOR oligonucleotide is included.

Example 51

[1130] Modified Backbones Improve Performance of ARRESTOROligonucleotides all Natural “ARRESTOR” Oligo with No 3′-Amine

[1131] The reactions described in the previous two Examples usedARRESTOR oligonucleotides that were constructed using 2′ O-methyl ribosebackbone, and that included a positively charged amine group on the 3′terminal nucleotide. The modifications were made specifically to reduceenzyme interaction with the primary probe/ARRESTOR oligonucleotidecomplex. During the development of the present invention, it wasdetermined that the 2′ O-methy modified oligonucleotides are somewhatresistant to cleavage by the 5′ nucleases, just as they are slowlydegraded by nucleases when used in antisense applications (See e.g.,Kawasaki et al., J. Med. Chem., 36:831 [1993]).

[1132] Further, as demonstrated in Example 35, the presence of an aminogroup on the 3′ end of an oligonucleotide reduces its ability to directinvasive cleavage. To reduce the possibility that the ARRESTORoligonucleotide would form a cleavage structure in this way, an aminogroup was included in the design of the experiments described in thisand other Examples.

[1133] Initial designs of the ARRESTOR oligonucleotides (sometimesreferred to as “blockers”) did not include these modifications, andthese molecules were found to provide no benefit in reducing backgroundcleavage in the sequential invasive cleavage assay and, in fact,sometimes contributed to background by inducing cleavage at anunanticipated site, presumably by providing some element to analternative cleavage structure. The effects of natural and modifiedARRESTOR oligonucleotide on the background noise in these reactions areexamined in this Example.

[1134] The efficacy of an “all-natural ARRESTOR oligonucleotide (i.e.,an ARRESTOR oligonucleotide that did not contain any base analogs ormodifications) was examined by comparison to an identical reactions thatlacked ARRESTOR oligonucleotide. All reactions were performed induplicate, and were conducted as follows. Two master mixes wereassembled, each containing 12.5 mM MOPS, pH 7.5, 500 fmoles primaryINVADER oligonucleotide #218-55-05 (SEQ ID NO: 171), 10 ng human genomicDNA (Novagen) and 30 ng AfuFEN1 enzyme for every 8 μl of mix. Mix Acontained no added HBV genomic amplicon DNA, mix B contained 600,000molecules of HBV genomic amplicon DNA, pAM6 #2. The mixes weredistributed to the reaction tubes, in aliquots of 8 μl/tube as follows:mix A to tubes 1, 2, 5 and 6; and mix B to tubes 3, 4, 7 and 8. Thesamples were incubated at 95° C. for 4 minutes to denature the HBVgenomic amplicon DNA. The reactions were then cooled to 67° C. and 2 ulof a mix containing 37.5 mM MgCl₂ and 10 pmoles 218-55-02B (SEQ ID NO:185) for every 2 μl, was added to each sample. The samples were thenincubated at 67° C. for 30 minutes. Two secondary reaction master mixeswere prepared, each containing 10 pmoles of secondary probe oligo#228-48-04N (SEQ ID NO: 178) and 2.5 pmoles of secondary targetoligonucleotide #218-95-04 (SEQ ID NO: 172) for every 3 μl of mix. Mix2A contained no additional oligonucleotide, while mix 2B contained 50pmoles of the natural ”ARRESTOR” oligonucleotide #241-62-02 (SEQ ID NO:186). After the initial 30 minute incubation at 67° C., the temperaturewas adjusted to 52° C., and 3 μl of a secondary reaction mix was addedto each sample, as follows: Mix 2A was added to samples #1-4; and Mix 2Bwas added to samples #5-8. The samples were then incubated for 30minutes at 52° C. The reactions were then stopped by the addition of 10μl of a solution of 95% formamide, 10 mM EDTA and 0.02% crystal violet.

[1135] All of the samples were heated to 95° C. for 2 minutes, and 4 μlof each sample were resolved by electrophoresis through a 20% denaturingacrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing45 mM Tris-Borate (pH8.3) and 1.4 mM EDTA. The results were imaged usingthe Molecular Dynamics Fluoroimager 595, excitation 488, emission 530.The resulting image is shown in FIG. 109A.

[1136] To compare the effects of the various modifications made to theARRESTOR oligonucleotides, reactions were performed using ARRESTORoligonucleotides having all natural bases, but including a 3′ terminalamine; ARRESTOR oligonucleotide having the 3′ portion composed of 2′O-methyl nucleotides, plus the 3′ terminal amine; and ARRESTORoligonucleotide composed entirely of 2′ O-methyl nucleotides, plus the3′ terminal amine. These were compared to reactions performed without anARRESTOR oligonucleotide. The reactions were conducted as follows. Twomaster mixes were assembled, all mixes contained 14.3 mM MOPS, pH 7.5,500 fmoles primary INVADER oligo #218-55-05 (SEQ ID NO: 171) and 10 nghuman genomic DNA (Novagen) for every 7 μl of mix. Mix A contained noadded HBV genomic amplicon DNA, mix B contained 600,000 molecules of HBVgenomic amplicon DNA, pAM6 #2. The mixes were distributed to thereaction tubes, at 7 μl/tube: mix A to tubes 1, 2, 5, 6, 9, 10, 13 and14; and mix B to tubes 3, 4, 7, 8, 11, 12, 15 and 16. The samples werewarmed to 95° C. for 4 minutes to denature the HBV DNA. The reactionswere then cooled to 67° C. and 3 μl of a mix containing 25 mM MgCl₂, 25pmoles 218-55-02B (SEQ ID NO: 185) and 30 ng AfuFEN1 enzyme per 3 μl,were added to each sample. The samples were then incubated at 67° C. for30 minutes. Four secondary reaction master mixes were prepared; allmixes contained 10 pmoles of secondary probe oligonucleotide #228-48-04B(SEQ ID NO: 190) and 2.5 pmoles of secondary target oligonucleotide#218-95-04 (SEQ ID NO: 172) for every 3 μl of mix. Mix 2A contained noadditional oligonucleotide, while mix 2B contained 100 pmoles of thenatural+amine ARRESTOR oligonucleotide #241-62-01 (SEQ ID NO: 187), mix2C contained 100 pmoles of partially O-methyl+amine oligonucleotide#241-62-03 (SEQ ID NO: 188) and mix 2D contained 100 pmoles of allO-methyl+amine oligonucleotide #241-64-01 (SEQ ID NO: 189). After theinitial 30 minute incubation at 67° C., the temperature was adjusted to52° C. and 3 μl of a secondary reaction mix was added to each sample, asfollows: mix 2A was added to samples #1-4; mix 2B was added to samples#5-8; mix 2C was added to samples #9-12; and mix 2D was added to samples#13-16. The samples were incubated for 30 minutes at 52° C., thenstopped by the addition of 10 μl of a solution of 95% formamide, 10 mMNaEDTA, and 0.2% crystal violet.

[1137] All samples were heated to 95° C. for 2 minutes, and 4 μl of eachsample were resolved by electrophoresis through a 20% denaturingacrylamide gel (19:1 cross-linked) with 7 M urea, in a buffer containing45 mM Tris-Borate (pH8.3) and 1.4 mM EDTA. The results were imaged usingthe Molecular Dynamics Fluoroimager 595, excitation 488, emission 530.The resulting image is shown in FIG. 109B.

[1138] In FIG. 109A, the left hand panel shows the reactions that lackedan ARRESTOR oligonucleotide, while the right hand panel shows the datafrom reactions that included the all natural ARRESTOR oligonucleotide.The first two lanes of each panel are from no-target controls, thesecond set of lanes contained target. The products of cleavage arevisible in the bottom one/fourth of each panel. The position at whichthe specific reaction products should run is indicated by arrows on leftand right.

[1139] It can be seen by examination of these data, that the reactionsrun in the absence of ARRESTOR oligonucleotide show reproducible qualitybetween the replicates, and show significant cleavage only when targetis present. In contrast, the addition of another unmodifiedoligonucleotide into the reactions causes great variation between thereplicate lanes (e.g., lanes 5 and 6 were provided with the samereactants, but produced markedly different results). The introduction ofthe all natural ARRESTOR oligonucleotide produced, rather than reduced,background in these no-target lanes, and increased cleavage at othersites (i.e., the bands other that those indicated by the arrows flankingthe panels). For these reasons the modifications that are describedabove, the effects of which are shown on FIG. 109B, were incorporated.

[1140] The first 4 lanes of FIG. 109B show the products of duplicatereactions without an ARRESTOR oligonucleotide, plus or minus the HBVtarget (lanes 1, 2, and lanes 3, 4, respectively); The next 4 lanes, 5,6 and 7, 8 used a natural ARRESTOR oligonucleotide having a 3′ terminalamine; lanes 9, 10 and 11, 12 used the ARRESTOR oligonucleotide with a3′ portion composed of 2′ O-methyl nucleotides, and having a 3′ terminalamine; lanes 13, 14 and 15, 16 used the ARRESTOR oligonucleotidecomposed entirely of 2′ O-methyl nucleotides and having a 3′ terminalamine. The products of cleavage of the secondary probe are visible inthe lower one third of each panel.

[1141] Visual inspection of these data shows that the addition of the 3′terminal amine to the natural ARRESTOR oligonucleotide suppresses theaberrant cleavage seen in FIG. 109A, but this ARRESTOR oligonucleotidedoes not improve the performance of the reaction, as compared to theno-ARRESTOR oligonucleotide controls. In contrast, the use of the 2′O-methyl nucleotides in the body of the ARRESTOR oligonucleotide doesreduce background, whether partially or completely substituted. Toquantify the relative effects of these modifications, the fluorescencefrom each of the co-migrating product bands was measured, the signalsfrom the duplicate lanes were averaged and the “fold over background”was calculated for each reaction containing target nucleic acid.

[1142] When ARRESTOR oligonucleotide was omitted, the target specificsignal (lanes 3, 4) was 27-fold over the no target background; thenatural ARRESTOR oligonucleotide+amine gave a signal of 17-fold overbackground; the partial 2′ O-methyl+amine gave a signal of 47-fold overbackground; and the completely 2′ O-methyl+amine gave a signal of 33fold over background.

[1143] These Figures show that both modifications can have a beneficialeffect on the specificity of the multiple, sequential invasive cleavageassay. They also show that the use of the 2′ O-methyl substitutedbackbone, either partial or entire, markedly improves the specificity ofthese reactions. It is intended that in various embodiments of thepresent inventon, that any number of modifications that make either theARRESTOR oligonucleotide or the complex it forms with the primary targetresistant to nucleases will provide similar enhancement.

Example 52

[1144] Effect of ARRESTOR Oligonucleotide Length on Signal Enhancementin Multiple Sequential Invasive Cleavage Assays

[1145] As noted in the Description of the Invention, the optimal lengthfor an ARRESTOR oligonucleotide depends upon the design of the othernucleic acid elements of the INVADER reaction, particularly on thedesign of the primary probe. In this Example, the effects of varying thelength of the ARRESTOR oligonucleotide were explored in systems usingtwo different secondary probes. A schematic diagram showing theseARRESTOR oligonucleotides aligned as they would hybridize to the primaryprobe oligonucleotide is provided in FIG. 110C. In this Figure, theregion of the primary probe that recognizes the target nucleic acid isshown underlined; the non-underlined portion, plus the first underlinedbase is the portion that is released by the first cleavage, and goes onto participate in the second or subsequent cleavage structure.

[1146] All reactions were performed in duplicate. The INVADER reactionswere done in a final volume of 10 μl final volume containing 10 mM MOPS,pH 7.5, mM MgCl₂, 500 fmoles of primary INVADER 241-95-01, (SEQ ID NO:176), 25 pmoles of primary probe 241-95-02 (SEQ ID NO: 175), 30 ng ofAfuFEN1 enzyme, and 10 ng of human genomic DNA, and if included, 1amoles of HBV amplicon pAM 6 #2. MOPS, target DNA, and INVADERoligonucleotides were combined to a final volume of 7 μl. Samples wereheat denatured at 95° C. for 5 minutes, then cooled to 67° C. During the5 minute denaturation, MgCl₂, probe and enzyme were combined. Theprimary INVADER reactions were initiated by the addition of 3 μl ofMgCl₂, probe and enzyme mix, to the final concentrations indicatedabove. Reactions were incubated for 30 minutes at 67° C. The reactionwere then cooled to 52° C., and each primary INVADER reaction receivedthe following secondary reaction components in a total volume of 3 μl:2.5 pmoles secondary target 241-95-07 (SEQ ID NO: 177), 10 pmoles ofeither secondary probe 228-48-04 (SEQ ID NO: 173), or 228-48-04N (SEQ IDNO: 178) and 100 pmoles of an ARRESTOR oligonucleotide, either 241-95-03(SEQ ID NO: 179), 241-95-04 (SEQ ID NO: 180), 241-95-05 (SEQ ID NO: 181)or 241-95-06 (SEQ ID NO: 182). The ARRESTOR oligonucleotide were omittedfrom some reactions as controls for ARRESTOR oligonucleotide effects.

[1147] The reactions were incubated at 52° C. for 34 minutes, and werethen stopped by the addition of 10 μl of 95% formamide, 10 mM EDTA, and0.02% crystal violet. All samples were heated to 95° C. for 1 minute,and 4 μl of each sample were resolved by electrophoresis through 20%denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffercontaining 45 mM Tris-Borate (pH8.3) and 1.4 mM EDTA. The results wereimaged using the Molecular Dynamics Fluoroimager 595, excitation 488,emission 530. The resulting images for the reactions with the shorterand longer secondary probes are shown in FIGS. 110A and 110B,respectively.

[1148] In each Figure, the products of cleavage are visible as bands inthe bottom half of each lane. The first 4 lanes of each Figure show theproducts of duplicate reactions without an ARRESTOR oligonucleotide,plus or minus the HBV target (lanes sets 1 and 2 respectively); in thenext 4 lanes, sets 3 and 4 used the shortest ARRESTOR 241-95-03 (SEQ IDNO: 179); lanes 5 and 6 used 241-95-04 (SEQ ID NO: 180); lanes 7 and 8used 241-95-05 (SEQ ID NO: 181); and lanes 9 and 10 used 241-95-06 (SEQID NO: 182).

[1149] The principal background of concern is the band that appears inthe “no target” control lanes (odd numbers; this band co-migrates withthe target-specific signal near the bottom of each gel panel). Visualinspection shows that the shortest ARRESTOR oligonucleotide was theleast effective at suppressing this background, and that the efficacywas increased when the ARRESTOR oligonucleotide extended further intothe portion that participates in the subsequent cleavage reaction. Evenwith this difference in effect, it can be seen from these data thatthere is much latitude in the design of the ARRESTOR oligonucleotide.The choice of lengths will be influenced by the temperature at which thereaction making use of the ARRESTOR oligonucleotide is performed, thelengths of the duplexes formed between the primary probe and the target,the primary probe and the secondary target, and the relativeconcentrations of the different nucleic acid species in the reactions.

Example 53

[1150] Effect of ARRESTOR Oligonucleotide Concentration on SignalEnhancement in Multiple Sequential Invasive Cleavage Assays

[1151] In examining the effects of including ARRESTOR oligonucleotidesin these cleavage reactions, it was of interest to determine if theconcentration of the ARRESTOR oligonucleotide in excess of the primaryprobe concentration would have an effect on yields of eithernon-specific or specific signal, and if the length of the ARRESTORoligonucleotide would be a factor. These two variable were investigatedin the following Example.

[1152] All reactions were performed in duplicate. The primary INVADERreactions were done in a final volume of 10 μl and contained 10 mM MOPS,pH 7.5; 7.5 mM MgCl₂, 500 fmoles of primary INVADER 241-95-01 (SEQ IDNO: 176), 25 pmoles of primary probe 241-95-02 (SEQ ID NO: 175), 30 ngof AfuFEN1 enzyme, and 10 ng of human genomic DNA. Where included, thetarget DNA was 1 amole of HBV amplicon pAM 6 #2, as described above.MOPS, target and INVADER were combined to a final volume of 7 μl. Thesamples were heat denatured at 95° C. for 5 minutes, then cooled to 67°C. During the 5 minute denaturation, MgCl₂, probe and enzyme werecombined. The primary INVADER reactions were initiated by the additionof 3 μl of MgCl₂, probe and enzyme mix. The reactions were incubated for30 minutes at 67° C. The reactions were then cooled to 52° C. and eachprimary INVADER reaction received the following secondary reactioncomponents: 2.5 pmoles secondary target 241-95-07 (SEQ ID NO: 177), 10pmoles secondary probe 228-48-04 (SEQ ID NO: 173); and, if included, 50,100 or 200 pmoles of either ARRESTOR oligonucleotide 241-95-03 (SEQ IDNO: 179) or 241-95-05 (SEQ ID NO: 181), in a total volume of 3 μl.Reactions were then incubated at 52° C. for 35 minutes. Reactions werestopped by the addition of 10 μl of 95% formamide, 10 mM EDTA, and 0.02%crystal violet. All of the samples were heated to 95° C. for 1 minute,and 4 μl of each sample were resolved by electrophoresis through 20%denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffercontaining 45 mM Tris-Borate (pH8.3), and 1.4 mM EDTA. The results wereimaged using the Molecular Dynamics Fluoroimager 595, excitation 488,emission 530. The resulting images are shown as a composite image inFIG. 111.

[1153] Each of the duplicate reactions were loaded on the gel inadjacent lanes and are labeled with a single lane number. All oddnumbered lanes were no-target controls. Lanes 1 and 2 had no ARRESTORoligonucleotide added; lanes 3-8 show results from reactions containingthe shorter ARRESTOR oligonucleotide, 241-95-03 (SEQ ID NO: 179); lanes9-14 show results from reactions containing the longer ARRESTORoligonucleotide, 241-95-05 (SEQ ID NO: 181). The products of cleavagefrom the secondary reaction are visible in the bottom one third of eachpanel. Visual inspection of these data (i.e., comparison of the specificproducts to the background bands) shows that both ARRESTORoligonucleotides have some beneficial effect at all concentration.

[1154] To quantify the relative effects of ARRESTOR oligonucleotidelength and concentration, the fluorescence from each of the co-migratingproduct bands was measured, the signals from the duplicate lanes wereaveraged and the “fold over background” (signal+target/signal-target)was calculated for each reaction containing target nucleic acid. Thereaction lacking an ARRESTOR oligonucleotide yielded a signalapproximately 27-fold over background. Inclusion of the shorter ARRESTORoligonucleotide at 50, 100 or 200 pmoles produced products at 42, 51 and60-fold over background, respectively. This shows that while the shortarrestr at the lowest concentration seems to be less effective than thelonger ARRESTOR oligonucleotides (See, previous Example) this can becompensated for by increasing the concentration of ARRESTORoligonucleotide, and thereby the ARRESTOR oligonucleotide:primary proberatio.

[1155] In contrast, inclusion of the longer ARRESTOR oligonucleotide at50, 100 or 200 pmoles produced products at 60, 32 and 24 fold overbackground, respectively. At the lowest concentration, the efficacy ofthis longer ARRESTOR oligonucleotide relative to the shorter ARRESTORoligonucleotide is consistent with the previous Example. Increasing theconcentration, however, decreased the yield of specific product,suggesting a competition effect with some element of the secondarycleavage reaction.

[1156] These data show that the ARRESTOR oligonucleotides can be used toadvantage in a number of specific reaction designs. The choice ofconcentration will be influenced by the temperature at which thereaction making use of the ARRESTOR oligonucleotide is preformed, thelengths of the duplexes formed between the primary probe and the target,the primary probe and the secondary target, and between the primaryprobe and the ARRESTOR oligonucleotide.

[1157] Selection of oligonucleotides for target nucleic acids other thanthe HBV shown here, (e.g., oligonucleotide composition and length), andthe optimization of cleavage reaction conditions in accord with themodels provided here follow routine methods and common practice wellknown to those skilled in the methods of molecular biology.

[1158] Example 45 demonstrated that some enzymes require an overlapbetween an upstream INVADER oligonucleotide and a downstream probeoligonucleotide to create a cleavage structure (FIG. 100). It has alsobeen determined that the 3′ terminal nucleotide of the INVADERoligonucleotide need not be complementary to the target strand, even ifit is the only overlapping base in the INVADER oligonucleotide (e.g., aswith the HCMV probes shown in FIG. 103). The requirement for an overlapcan serve as a convenient basis for detecting single base polymorphisms(SNPs) or mutations in a nucleic acid sample.

[1159] For detection of single base variations, at least twooligonucleotides (e.g., a probe and an INVADER oligonucleotide)hybridize in tandem to the target nucleic acid to form the overlappingstructure recognized by the CLEAVASE enzyme to be used in the reaction.An unpaired “flap” is included on the 5′ end of the probe. The enzymerecognizes the overlap and cleaves off the unpaired flap, releasing itas a target-specific product. Enzymes that have a strong preference foran overlapping structure, i.e., that cleave the overlapping structure ata much greater rate than they cleave a non-overlapping structure includethe FEN-1 enzymes from Archaeoglobus fulgidus and Pyrococcus furiosusand such enzymes are particularly preferred in the detection ofmutations and SNPs. In the secondary reaction, the released flap servesas an INVADER oligonucleotide to create another overlapping cleavagestructure (e.g., as shown in FIG. 96). In the following examples, thereleased flap creates this overlapping structure in conjunction with aFRET cassette, a single oligonucleotide having a region ofself-complementarity to form a hairpin (FIG. 112A) When the FRETcassette is cleaved to release its 5′ nucleotide, the fluorescent dye(F) and the quencher (Q) on the cassette are separated and a detectablefluorescence signal is produced. If the probe and the target sequence donot match perfectly at the cleavage site (e.g., as in FIG. 112B), theoverlapped structure does not form, cleavage is suppressed, and nofluorescence will be produced.

[1160] The reactions may be performed under conditions in which theprobes and FRET cassettes turn over continuously without temperaturecycling or cleavage. When an uncut probe hybridizes to the target nextto an overlapping INVADER oligonucleotide, the probe can be cleaved toproduce a target-specific product, which in turn enables the cleavage ofmany FRET cassettes.

[1161] The following eight examples demonstrate the design andapplication of the sequential INVADER assay with FRET cassette detectionto analysis of SNPs and mutations in a variety of nucleic acid samples.

Example 54

[1162] Detection of Single Nucleotide Polymorphisms in the HumanApolipoprotein E Gene

[1163] This Example describes an assay for the detection of SNPs in thehuman Apolipoprotein E gene. Probe and INVADER oligonucleotides weredesigned to target single nucleotide polymorphisms (SNPs) at twopositions in the human apolipoprotein E (apoE) gene. Three differentalleles exist for the apoE gene, epsilon 2, epsilon 3, and epsilon 4,which code for 3 different isoforms of the apoE protein, termed E2, E3and E4. The different isoforms vary in at amino acid positions 112 or158 (see Table A), and each variation is caused by a single base changein the corresponding codon. TABLE A 112 158 ISOFORM codon amino acidcodon amino acid E2 TGC cys TGC cys E3 TGC cys CGC arg E4 CGC arg CGCarg

[1164] INVADER and probe oligonucleotides were designed using thealgorithm described above (detailed description of the invention), withthe probe set selected to operate at 63° C. As shown in FIGS. 113A-D,one INVADER oligonucleotide and two unlabelled, probe oligonucleotides,one for each variant at a given locus, were designed for each codon. Forcodon 112, the probes were designed to detect either the C nucleotide(to code for arginine; SEQ ID NO: 197) or a T nucleotide (to code forcysteine; SEQ ID NO: 198). For codon 158, the probes were designed todetect either a C nucleotide (to code for arginine; SEQ ID NO: 199) or aT nucleotide (to code for cysteine; SEQ ID NO: 200). A FRET cassette tobe used with all of the probe sets was also synthesized (SEQ ID NO: 201,FIG. 113). In this Example and in the following Examples, alloligonucleotides were synthesized using standard phosphoramiditechemistries. Primary probe oligonucleotides were unlabeled. The FRETcassettes were labeled by the incorporation of Cy3 phosphoramidite andfluorescein phosphoramidite (Glen Research). While designed for 5′terminal use, the Cy3 phosphoramidite has an additional monomethoxytrityl (MMT) group on the dye that can be removed to allow furthersynthetic chain extension, resulting in an internal label with the dyebridging a gap in the sugar-phosphate backbone of the oligonucleotide(as diagrammed in the panels of FIG. 113). While a nucleotide may beomitted at this position to accommodate the dye, we have determined itis not necessary, and no nucleotides were omitted from the FRETcassettes used in these examples. Amine or phosphate modifications,where indicated, were used on the 3′ ends of the primary probes and theFRET cassettes to prevent their use as invasive oligonucleotides.2′-O-methyl bases in the secondary target oligonucleotides are indicatedby underlining and were also used to minimize enzyme recognition of 3′ends. In addition, reactions having synthetic target DNAs were used aspositive controls to verify the activity of the reaction components. Thecontrol targets are illustrated in FIGS. 113A-D. The 112 arg, 112 cys,158 arg, and 158 cys control oligonucleotides are SEQ ID NOS: 191-194,respectively. Genomic DNA sample AG09714 was purchased from Coriell.This sample was quantitated via Pico Green and diluted with Tris with0.1 mM EDTA to a concentration of approximately 100 ng/μl. This sample(9714 in FIG. 114A) was used to test for the 112 mutation. Samples39634, 32435 and 31071 were purchased as whole blood from LampireBiological Labs., Inc. Samples 511, 537, 538 and 539 were whole bloodsamples donated by the Blood Center of Southeast Wisconsin (Milwaukee,Wis.). Genomic DNA was prepared via the PUREGENE Blood Kit (Gentra)according to the manufacturer's instructions. Samples 39634 and 32435were used to test for the 112 mutation, and sample 31071, 511, 537, 538and 539 were used to test for the 158 mutation. One μl of genomic DNAwas used per reaction, with 9 μl of water, for a final volume of 10 μl.Although determination of the full genotype for any one sample generallyrequires analysis of both loci, the samples listed above were selectedto show representative signals from, and thus the functioning of, eachprobe set. Complete genotyping requires each sample to be tested withboth probe sets.

[1165] The experiment comprised testing each genomic DNA for theindicated alleles, along with reactions having no target DNA to allowmeasurement of any background signal not attributable to the presence ofa target sequence. Reactions testing the 112 locus were done inquadruplicate, reactions testing the 158 locus were done in triplicate.

[1166] Reaction components were prepared as batch mixes for dispensingto the individual test reactions. Batches of INVADER mix for each allelewere prepared, comprising for each planned reaction: 4 μl of 16% PEG8000/50 mM MOPS pH 7.5 and 1 μl of 1 μM INVADER oligonucleotide (eitherthe 112 or the 158). Batches of CLEAVASE enzyme/Mg²⁺/probe mix for eachallele were prepared, comprising for each planned reaction: 2 μl of 75mM MgCl₂, 1 μl of 10 μM FRET cassette, 1 μl 10 μM probe oligonucleotide(any one of the 112 or the 158 T or C probe oligonucleotides), and 1 μlof 200 ng/μl Afu FEN-1 enzyme.

[1167] For each reaction, 5 μl of INVADER reaction mix were aliquotedinto each well of a 96-well Low Profile Polypropylene Microplate (MJResearch,). Ten μl of each control DNA or genomic sample (approximately100 ng-190 ng) were added and mixed by pipetting up and down. Theno-target controls received 1 μg of yeast tRNA instead of target DNA.The reactions comprising the synthetic targets as positive controlsincluded either 150 or 100 zeptomoles (zmoles) of the 112 or 158synthetic targets, respectively, and 1 μg of yeast tRNA. Each reactionwas overlaid with 20μl of clear CHILLOUT liquid wax, and incubated at95° C. for 5 minutes. The reaction temperature was then lowered to 63°C., 5 μl of the appropriate CLEAVASE enzyme/Mg²⁺/Probe reaction mix wasadded to each reaction and mixed by pipetting up and down 3-5 times, andthe reactions were further incubated for 4 hours at 63° C., and wereread directly on a CYTOFLUOR Series 4000 Fluorescence Multi-well PlateReader, (PerSeptive Biosystems), using the following settings:Excitation (wavelength/bandwidth): 485/20 nm; Emission(wavelength/bandwidth): 530/25 nm; Gain: 37. The net averagedfluorescence signal was calculated by subtracting the averaged no-targetsignal (background) from the corresponding averaged target DNA reactionsignal and the data were plotted using Excel spreadsheet software(Microsoft).

[1168] Results for the ApoE 112 and ApoE 158 loci are shown graphicallyin FIGS. 114A and 114B, respectively, with target DNAs indicated on thehorizontal axis and the fluorescence units indicated on the verticalaxis. The white bars represent the net averaged signal from the “C”probe, while the dark bars represent the net averaged signal from the“T” probe. The reactions having synthetic targets are indicated as Syn Cand Syn T for the C and T controls, respectively. At both loci, thesamples that are homozygous for either the C or T allele are easilyidentified by having a strong signal from only one probe or the other,while the heterozygous samples, are easily identified by having strongsignals from both the C and T probes.

Example 55

[1169] Detection of Mutations in the Human Hemochromatosis (HFE) Gene

[1170] The human hemochromatosis (HFE gene) gene is located in the MHCregion of chromosome 6, and was initially named HLA-H. It was laterrenamed the HFE gene, in accordance with the WHO Nomenclature Committeefor Factors of the HLA System. Two different, single-base variations inthe HFE gene are responsible for the vast majority of the cases ofhereditary hemochromatosis, or iron overload disease. The most commonvariant, termed C282Y, is caused by a change from the wild type (WT)adenine (A) to the mutant (MT) guanine (G) at codon 282 that causes anamino acid change from a cysteine to a tyrosine residue. The secondvariation commonly detected in individuals suffering from iron overloaddisorder is termed H63D, and is caused by a WT cytosine (C) to a MTguanine (G) change at codon 63 that causes an amino acid change from ahistidine to an aspartic acid residue in the expressed protein.

[1171] INVADER and probe oligonucleotide sets were designed as describedabove to detect both the WT and MT alleles for both the C282Y and H63Dsites and are shown in FIGS. 115A-D. Detection of the C282 polymorphismused one INVADER oligonucleotide (SEQ ID NO: 202), one probe specificfor each variant of the allele (WT and MT, SEQ ID NOS: 208 and 209,respectively) and a first FRET cassette (SEQ ID NO: 210).Oligonucleotides for the H63 locus included an INVADER oligonucleotide(SEQ ID NO: 203), one probe specific for each variant of the allele (WTand MT, SEQ ID NOS: 211 and 212, respectively), and a second FRETcassette (SEQ ID NO: 213). In addition, reactions having synthetictarget DNAs were used as positive controls to verify the activity of thereaction components. The control targets are illustrated in FIGS.115A-D. The C282 WT, C282 MT, H63 WT and H63 MT control oligonucleotidesare SEQ ID NOS: 204-207, respectively. Human genomic DNA samples 14640,14641, 14646, 14690, and 14691 were purchased from Coriell CellRepositories ( Catalog #s NA14640, NA14641, NA1446, NA14690, andNA14691).

[1172] Reactions were performed and analyzed as described in Example 54.The reactions comprising the synthetic targets as positive controlsincluded 100 zmoles of the synthetic target and 1 μg of yeast tRNA.

[1173] Results are shown graphically in FIG. 116, with target DNAsindicated on the horizontal axis and the fluorescence units indicated onthe vertical axis. The white bars represent the net signal from thewild-type probe, while the dark bars represent the net signal from themutant probe. The left half of the graph indicates samples tested forthe C282Y polymorphism, while the right half of the graph indicatessamples tested for the H63D polymorphism. No target controls areindicated as “NT” and the synthetic targets are indicated as SynWT andSynMT for wild-type and mutant controls, respectively. At both loci, thesamples that are homozygous for either WT or MT are easily identified byhaving a strong signal from only one probe or the other, while theheterozygous samples, are easily identified by having strong signalsfrom both the WT and MT probes.

Example 56

[1174] Detection of Mutations in the Human MTHFR

[1175] Human 5,10-methylene-tetrahydrofolate reductase (MTHFR) is amajor enzyme in the folate-dependent regulation of methionine andhomocysteine concentrations. The wild-type protein plays a critical rolein the conversion of homocysteine to methionine. A particular variationin the MTHFR protein, termed C677T and caused by a C to T transition inthe MTHFR gene, has been correlated with myriad diseases and defects,including cardiovascular and neurological disorders. This Exampledescribes an assay for the detection of the WT and MT alleles for theMTHFR 677 SNP.

[1176] INVADER and probe oligonucleotide sets were designed as describedabove to detect both the WT and MT allele for the MTHFR 677 site(INVADER, WT probe, MT probe, and FRET cassette are SEQ ID NOS: 216,217, 218 and 225, respectively). Positive control targets weresynthesized for the WT and MT alleles at the 677 site (SEQ ID NOS: 214and 215, respectively); oligonucleotides are shown in FIGS. 117A and117B.

[1177] Human genomic DNA samples 01532 and 01560 were purchased fromCoriell. These samples had been characterized by “PCR” at Coriell forthe MTHFR genotype. They were also characterized in house by PCR/RFLPanalysis for genotype confirmation. Human genomic sample 32435 waspurchased as whole blood from Lampire Biological Labs., Inc.(Coopersberg, Pa.), and genomic DNA was prepared via the Gentra PUREGENEBlood Kit (Minneapolis, Minn.) according to the manufacturer'sinstructions. Samples were quantitated via PicoGreen (Molecular Probes,Eugene Oreg.) and diluted with TE to a concentration of approximately 10ng/μl. 100 ng (10 μl) of each sample was used in each reaction.

[1178] Triplicate reactions were performed and analyzed as described inExample 54, except the INVADER mixes contained 1 μl of 0.5 μM INVADERoligonucleotide for each reaction. The reactions comprising thesynthetic targets as positive controls included 50 zmoles of thesynthetic target and 1 μg of yeast tRNA. Reactions simulating aheterozygous sample included 50 zmoles of each control target.

[1179] Results are shown graphically in FIG. 118, with target DNAsindicated on the horizontal axis and the fluorescence units indicated onthe vertical axis. The white bars represent the net averaged signal fromthe wild-type probe, while the dark bars represent the net averagedsignal from the mutant probe. The synthetic targets are indicated asSynWT and SynMT for wild-type and mutant controls, respectively andSynHET for a mixture of the two to simulate a heterozygous sample. Atboth loci, the samples that are homozygous for either WT or MT areeasily identified by having a strong signal from only one probe or theother, while the heterozygous samples are easily identified by havingstrong signals from both the WT and MT probes.

Example 57

[1180] Detection of Mutations in the Human Prothrombin (Factor II) Gene

[1181] The prothrombin A20210G mutation has been determined to be a riskfactor for thromboembolism. The mutation occurs in the 3′ untranslatedregion of the prothrombin gene, replacing the WT adenine (A) at position20210 with the MT guanine (G). This Example describes an assay for thedetection of the WT and MT alleles of the prothrombin A20210G mutation

[1182] INVADER and probe oligonucleotide sets were designed as describedabove to detect both the WT and MT alleles at the prothrombin 20210 site(INVADER, WT probe, MT probe and FRET cassette are SEQ ID NOS: 222-225,respectively). Positive control targets were synthesized for the WT andMT alleles at the 20210 site (SEQ ID NOS: 220 and 221, respectively);oligonucleotides are shown in FIGS. 119A and 119B.

[1183] Three patient samples of human genomic DNA were donated by theBlood Center of Southeast Wisconsin, identified as 2196, 2263 and 2265.These samples were purified at the Blood Center via QIAGEN BioRobot 9600(QIAGEN #900200), quantitated by Pico Green (Molecular Probes) and werefound to be at a concentration of 30-50 ng/ul.

[1184] Duplicate reactions were performed and analyzed as described inExample 54, except the INVADER mixes contained 1 μl of 0.5 μM INVADERoligonucleotide for each reaction, and the mix was diluted with 1 volumeof dH₂O (i.e., 5 μl per reaction) so that the INVADER mix was dispensed10 μl aliquots, while the DNA was dispensed in 5 μl aliquots, adding150-250 ng of genomic DNA per reaction. Each no target control reactionreceived 1 μg of yeast tRNA, and the reactions comprising the synthetictargets as positive controls included 300 zmoles of WT control or 50zmoles of MT control and 1 μg of yeast tRNA.

[1185] Results are shown graphically in FIG. 120, with target DNAsindicated on the horizontal axis and the fluorescence units indicated onthe vertical axis. The white bars represent the net averaged signal fromthe wild-type probe, while the dark bars represent the net averagedsignal from the mutant probe. Synthetic targets are indicated as SynWTand SynMT for wild-type and mutant controls, respectively. Samples thatare homozygous for the WT are easily identified by having a strongsignal from only the WT probe, while the heterozygous sample is easilyidentified by having strong signals from both the WT and MT probes.

Example 58

[1186] Detection of the HR-2 Mutation in the Human Factor V Gene

[1187] This Example describes an assay for the deteciton of the R-2polymorphism in the human factor V gene. The R-2 polymorphism in thehuman factor V gene is located in exon 13 of the factor V gene, and isthe result of an A to G transition at base 4070, replacing the wild-typeamino acid histidine with the mutant arginine in the mature protein. TheR-2 polymorphism is one of a set of mutations termed collectively HR-2.The HR-2 haplotype is defined by 6 nucleotide base substitutions inexons 13 and 16 of the factor V gene, and is associated with anincreased functional resistance to activated protein C both in normalsubjects and in thrombophilic patients. When present as a compoundheterozygote in conjunction with the Leiden mutation (see Example 60,below), clinical symptoms are comparable to those seen in patientshomozygous for the Leiden mutation.

[1188] Within about a 600-base pair region surrounding the R2 allele,four sub-regions of DNA, each encompassing the sequence of the WTINVADER probe set, (approximately 22 bases in length), contain sequencesimilar to that immediately surrounding the R2 allele. These repeatedsequences can be detected by an INVADER and probe oligonucleotide setdesigned for the wild-type R-2 sequence. Because of the repeatedsequence, reactions with this INVADER/probe set yield very high signal,even with genomic samples containing R-2 mutants, thus greatlyincreasing the risk of misinterpreting the data. In the example of anR-2 heterozygote, the signal generated by the R-2 mutant would beextremely low compared to the wild-type signal. It is thus possible thatone might err and interpret the data as wild-type, not as heterozygous.The same would hold true even for a homozygous mutant R-2 sample.Therefore, instead of having an INVADER/probe set to detect the WT R-2allele, an INVADER/probe set was developed to detect sequences in thesingle copy α-actin gene, thus providing an internal reaction control,as well as an internal signal intensity control. Since the α-actin geneis single copy, the signal levels generated in the detection of thissequence will be comparable to that generated in the detection of theR-2 mutant allele, and the probability of incorrect data interpretationdue to WT signal overwhelming that generated by the MT is no longer anissue.

[1189] In the previous examples, each sample was assayed in twodifferent reactions, one reaction tested for the presence of wild-typesequence and one reaction tested for the presence of mutant sequence. Inthis example, each sample is tested with both the α-actin internalcontrol and the MT R-2 INVADER/probe sets. INVADER and probeoligonucleotide sets were designed as described above to detect both theMT R-2 and α-actin control sequence (MT R-2 INVADER and probe, and theFRET cassette are SEQ ID NOS: 228, 229 and 230, respectively; mactinINVADER and probe are SEQ ID NOS: 231 and 232, respectively). Positivecontrol targets were synthesized for the Mutant R-2 allele and theα-actin gene (SEQ ID NOS: 226 and 227, respectively); oligonucleotidesare shown in FIG. 121A and B.

[1190] Human genomic DNA sample 39021 was obtained from Sigma (Catalog #D6537) and uncharacterized human genomic sample 15506 was obtained fromCoriell(Catalog # NA15506, Camden, N.J. 08103) Samples were diluted to10 ng/μl with Tris-EDTA, pH 8.0. Ten μl (100 ng) was used per reaction.No target controls received 1 μg of yeast tRNA instead of human genomicDNA. Reactions were performed and analyzed as described in Example 54,except the INVADER mixes contained 0.5 μl each of 2 μM R-2 INVADERoligonucleotide and 2.0 μM α-actin INVADER oligonucleotide. The probemaster mixes contained 1 μl of either the 10 μM R-2 probe or 1 μl of 10μM α-actin probe, 2 μl of 75 mM MgCl₂, 1 μl of 10 μM FRET cassette and 1μl 200 ng/μl Afu FEN-1 enzyme per reaction. The reactions comprising thesynthetic targets as positive controls included 100 zmoles of thesynthetic target and 1 μg of yeast tRNA. The SynHET and SynMT reactionscontained mixtures of synthetic targets at 2:1 and 1:1 of theα-actin:R-2 mutant targets, respectively.

[1191] Reactions were read directly on a CYTOFLUOR Multi-well PlateReader Series 4000 (PerSeptive Biosystems) using the followingparameters: Excitation wavelength/bandwidth 485 nm/20 nm, Emissionwavelength/bandwidth 530 nm/25 nm, gain 36.

[1192] Results are shown graphically in FIG. 122, with target DNAsindicated on the horizontal axis and the fluorescence units indicated onthe vertical axis. The white bars represent the net signal from theinternal control probe, while the dark bars represent the net signalfrom the R-2 mutant probe. Synthetic targets are indicated as SynIC andSynMT for internal control and mutant R-2 controls, respectively andSynHET for a mixture of the two to simulate a sample that isheterozygous at the R-2 allele. The sample that does not have the MT R-2allele is easily identified by having a strong signal from only the ICprobe, while that which is heterozygous at the R-2 allele is identifiedby having signals from both the IC and the MT R-2 probes, but at a rationear 2:1. A sample homozygous for the mutation at the R-2 allele (notshown) would show nearly equal signal from each probe, as shown with theSynMT control.

Example 59

[1193] Detection of Single Nucleotide Polymorphisms in the Human TNF-αGene

[1194] The human cytokine tumor necrosis factor α (TNF-α) gene has beenshown to be a major factor in graft rejection; the more TNF-α present inthe system, the greater the rejection response to transplanted tissue.The mutation detected in this example is located in the promoter regionof the TNF-α gene at position −308 (minus 308). The WT guanine (G) isreplaced with a MT adenine (A). This result of this promoter mutation isthe enhancement of transcription of TNF-α by 6 to 7 fold. This Exampledescribes an assay for the detection of the −308 mutation in thepromoter of the human TNF-α gene.

[1195] INVADER and probe oligonucleotide sets were designed as describedabove to detect both the WT and MT alleles at the −308 site (INVADER, WTprobe, and MT probe are SEQ ID NOS: 235, 236 and 237, respectively; FRETcassette is SEQ ID NO: 225). Positive control targets were synthesizedfor the WT and MT alleles at the −308 site (SEQ ID NOS: 233 and 234,respectively); oligonucleotides are shown in FIGS. 123A and 123B.

[1196] Purified human genomic DNA samples (M2, M3 and M4) were donatedby the Mayo Clinic (Rochester Minn.).

[1197] Triplicate reactions were performed as described in Example 54,except they were stopped after the four hour incubation at 63° C. by theaddition 100 μl of 100 mM EDTA. Reactions comprising the synthetictargets as positive controls included 100 zmoles of the synthetic targetand 1 μg of yeast tRNA. No target controls received 1 μg of yeast tRNAinstead of human genomic DNA. 100 μl of each stopped reaction wastransferred to a Nunc 96 well Maxisorb plate (VWR Scientific) and readon a CYTOFLUOR Multi-well Plate Reader Series 4000 (PerSeptiveBiosystems) using the following parameters: Excitationwavelength/bandwidth 485 nm/20 nm, Emission wavelength/bandwidth 530nm/25 nm; gain 65.

[1198] Results are shown graphically in FIG. 124, with target DNAsindicated on the horizontal axis and the fluorescence units indicated onthe vertical axis. The white bars represent the net averaged signal fromthe wild-type probe, while the dark bars represent the net averagedsignal from the mutant probe. The synthetic targets are indicated asSynWT and SynMT for wild-type and mutant controls, respectively andSynHET for a mixture of the two to simulate a heterozygous sample. Thesamples that are homozygous for either WT or MT are easily identified byhaving a strong signal from only one probe or the other, while theheterozygous sample is easily identified by having signals from both theWT and MT probes.

Example 60

[1199] Detection of the Factor V Leiden Mutation

[1200] The “Leiden ” mutation in blood coagulation factor V results froma cytosine “C” to a thymidine “T” base change in exon 1 of the factor Vgene. The mutant protein has glutamine at amino acid position 506,instead of the wild-type arginine. This substitution prevents activatedprotein C from cleaving and inactivating factor V. The active form ofthe protein therefore remains abundant in the blood stream and continuesto promote coagulation. This Example describes an assay for thedetection of the Leiden mutation of the human factor V gene.

[1201] INVADER and probe oligonucleotide sets were designed as describedabove to detect both the WT and MT alleles at the 506 site (INVADER, WTprobe, and MT probe are SEQ ID NOS: 240, 241 and 242, respectively; theFRET cassette is SEQ ID NO: 225). Positive control targets weresynthesized for the WT and MT alleles at the 506 site (SEQ ID NOS: 238and 239, respectively); oligonucleotides are shown in FIGS. 125A and125B.

[1202] Whole blood samples were obtained from the Midwest HemostasisCenter (Muncie, Ind.). Samples were characterized for the Factor VLeiden genotype by the Midwest Hemostasis Center via methods involvingPCR. Buffy coats were isolated as previously described, and genomic DNAwas purified using the QIAamp 96 DNA Blood Kit (Qiagen, Valencia)according to the manufacturer's instructions, except that the sampleswere eluted in 200 μl of elution buffer. The purified DNA samples werequantitated by Pico Green (Molecular Probes) and were then diluted withTE to a concentration of 15-60 ng per μl. Single reactions wereperformed as described in Example 54. The no-target control reactionreceived 1 μg of yeast tRNA instead of DNA, and the reactions comprisingthe synthetic targets as positive controls included 200 zmoles of thesynthetic target and 1 μg of yeast tRNA.

[1203] After the 4 hour incubation at 63° C., reactions were readdirectly on a CYTOFLUOR Multi-well Plate Reader Series 4000 (PerSeptiveBiosystems) using the following parameters: Excitationwavelength/bandwidth 485 nm/20 nm, Emission wavelength/bandwidth 530nm/25 nm, gain 65.

[1204] Results are shown graphically in FIG. 126, with target DNAsindicated on the horizontal axis and the fluorescence units indicated onthe vertical axis. The white bars represent the net averaged signal fromthe wild-type probe, while the dark bars represent the net averagedsignal from the mutant probe. The synthetic targets are indicated asSynWT and SynMT for wild-type and mutant controls, respectively andSynHET for a mixture of the two to simulate a heterozygous sample. Thesamples that are homozygous for either WT or MT are easily identified byhaving a strong signal from only one probe or the other, while theheterozygous samples are easily identified by having signals from boththe WT and MT probes.

Example 61

[1205] Detection of Methicillin-Resistant Staphalococcus aureus

[1206]Staphylococcus aurcus is recognized as one of the major causes ofinfections in humans occurring both in the hospital and in the communityat large. One of the most serious concerns in treating any bacterialinfection is the increasing resistance to antibiotics. The growingincidence of methicillin-resistant S. aureus (MRSA) infections worldwidehas underscored the importance of both early detection of the infectiveagent, and defining a resistance profile such that proper treatment canbe given. The primary mechanism for resistance to methicillin involvesthe production of a protein called PBP2a, encoded by the mecA gene. ThemecA gene is not native to Staphalococcus aureus, but is ofextra-species origin. The mecA gene is, however, indicative ofmethicillin resistance and is used as a marker for the detection ofresistant bacteria. To identify methicillin resistant S. aureus vianucleic acid techniques, both the mecA gene and at least one speciesspecific gene must be targeted. A particular species-specific gene, thenuclease or nuc gene is used in the following example. This Exampledescribes an assay for the detection of MRSA.

[1207] INVADER and probe oligonucleotide sets were designed as describedabove to detect both the mecA gene and the nuc gene (meA INVADER andprobe, are SEQ ID NOS: 243 and 244, respectively; nuc INVADER and probeare SEQ ID NOS: 246 and 247, respectively; the FRET cassette for bothINVADER/probe sets is SEQ ID NO: 245); oligonucleotides are shown inFIG. 127A and 127B. The mecA and nuc target sequences shown are SEQ IDNOS: 252 and 253, respectively. Samples of methicillin-resistantStaphalococcus aureus were purchased from American Type CultureCollection (ATCC., Catalog #33591). Samples of methicillin sensitiveStaphalococcus aureus (MSSA) were obtained from Gene Trak, Inc. (GT#2431), and samples of Staphalococcus haemolyticus were obtained fromATCC (ATCC #29970). The bacterial samples were streaked onto standardblood agar plates and grown at 37° C. for 14-18 hours. DNA samples fromMRSA, MSSA, and S. haemolyticus were prepared as follows. A singlecolony was suspended in 50 μl of 10 mM TRIS pH 7.5 in a 1.5 ml microfugetube. The sample was incubated at 65° C. for 5 minutes, and thenmicro-waved on the highest setting for 4 minutes. Ten μl of thispreparation was used in each reaction.

[1208] Positive controls were created by polymerase chain reaction. A533 base-pair DNA fragment of the mecA sequence and a 467 base pair DNAfragment of the nuc gene sequence were amplified and isolated asfollows. PCR primer sequences used for mecA gene amplification were5′-AAA ATC GAT GGT AAA GGT TGG C-3” (SEQ ID NO: 248)and 5′-AGT TCT GCAGTA CCG GAT TTG C-3′ (SEQ ID NO: 249). PCR primer sequences used for nucgene sequence amplification were 5′-TCGCTACTAGTTGCTTAGTG-3′ (SEQ ID NO:250) and 5′-GTAAACATAAGCAACTTTAG-3′ (SEQ ID NO: 251). MRSA and MSSAtarget DNA was isolated as described above. PCR reactions were doneusing the AMPLITAQ DNA Polymerase Kit with GENEAMP (PE CorporationCatalog # N808-0152). Separate reactions were done for the mecA and nucsequences, and were performed in a 100 μl final volume containing thefollowing components: 10 μl of 10×PCR buffer, 2.5 μl of 10 μM upstreamprimer and downstream primer, 2 μl of 10 mM dNTP mix, 1.0 μl AMPLITAQDNA polymerase, 2 μl (10-50 ng) of bacterial DNA (the MRSA or the MSSA),and 80 μl of water for a final volume of 100 μl. Reactions were coveredwith approximately 50 μl of CHILLOUT liquid wax (MJ Research) and cycledas follows: the mecA reactions were denatured at 97° C. for 3 minutes.Reactions were then cycled at 97° C. for 1 minute, 52° C. for 30seconds, 72° C. for 1 minute. This was repeated 5 times After the final72° C. 1 minute incubation, reactions were again heated to 94° C. for 30seconds, 52° C. for 30 seconds and 72° C. for 1 minute, for 30 cycles.mecA reactions were then incubated at 72° C. for 7 minutes, then held at4° C. until purification.

[1209] The nuc amplification reactions were denatured at 97° C. for 3minutes. Reactions were then cycled at 97° C. for 1 minute, 48° C. for30 seconds, 72° C. for 1 minute. This was repeated for 5 cycles. Afterthe final 72° C., 1 minute incubation, reactions were heated to 94° C.for 30 seconds, 48° C. for 30 seconds and 72° C. for 1 minute. This wasrepeated for 30 cycles. Reactions were then incubated at 72° C. for 7minutes, and finally cooled to 4° C. and held until purification.

[1210] After amplification, reactions were run on a 1% agarose gel in 1%TBE buffer with 50-2000 base-pair markers (Novagen,Cat #69278-3); bandswere visualized via ethidium bromide staining followed by ultravioletillumination. The appropriately sized bands were excised from the geland purified by the QIAquick Gel Extraction Kit (QiagenCat #28704)according to the manufacturer's protocol. Each column was eluted twicewith 50 μl of elution buffer. The concentration of the purified PCRsynthetic target DNA was determined by OD₂₆₀, and diluted to a stockconcentration of 50 fmoles per microliter. Positive controls were usedat a concentration of 10 amole per reaction with a 10 μl addition.Control reactions with no target were also performed, using humangenomic DNA at 10 ng/μl in place of a bacterial sample. All samples wereadded in a volume of 10 μl.

[1211] INVADER reactions were performed in MJ 96 well Low ProfilePolypropylene Microplates (MJ Research MLL-9601) in duplicate in a finalvolume of 15 μl. An INVADER reaction master mix was prepared andcontained (per reaction) 1.5 μl water, 2.5 μl 100 mM MOPS, 5 μl of 20%PEG (8000MW), 0.5 μl of 1 μM nuc INVADER oligonucleotide and 0.5 μl ofmecA INVADER oligonucleotide . Five μl of the INVADER master mix wereadded to each sample and control well. 10 μl of the target bacterial DNAsamples, prepared as described above, or 10 μl (10 amoles) of positivecontrol target, or 10 μl (100 ng of human genomic DNA) of the no targetcontrol sample were added to each well. Samples were overlaid with 15 μlclear Chill-out wax (MJ Research,) and incubated at 95° C. for 5 minutesin an MJ Research Thermocycler with Hot Bonnet. Two different probemaster mixes were prepared, one containing the mecA probeoligonucleotide, one containing the nuc probe oligonucleotide. The probemaster mixes contained (per reaction) 2 μl of 93.75 mM MgCl₂, 1 μl of10μM FRET oligonucleotide, 1 μl of either 10 μM mecA probeoligonucleotide or 10 μM nuc probe oligonucleotide, 1 μl of 100 ng/μlAfu FEN-1 enzyme (Third Wave Technologies, Cat #96004D). The temperaturewas cooled to 64° C. and 5 μl of the appropriate probe master mix (suchthat each control or sample reaction is tested with both the mecA andthe nuc probe) was added below the Chill-out wax layer to each well andmixed by pipetting up and down 5 times. The plate was covered withMICROSEAL ‘A’ Film (MJ Research,) and incubated at 64° C. for 30minutes. After the 30 minute incubation, reactions were cooled to roomtemperature, placed on a Perkin Elmer MICROAMP base (cat# N801-0531) andread on a CYTOFLUOR Multi-well Plate Reader Series 4000 (PerSeptiveBiosystems) using the following parameters: Excitationwavelength/bandwidth 485 nm/20 nm, Emission wavelength/bandwidth 530nm/25 nm, gain 40, 30 reads per well, temperature 25° C.

[1212] Results are shown graphically in FIG. 128, with target DNAsindicated on the horizontal axis and the fluorescence units indicated onthe vertical axis. The white bars represent the net averaged signal fromthe mecA probe, while the dark bars represent the net averaged signalfrom the nuc probe.

[1213] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described methods and systems of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the relevant fields are intended to be within the scopeof the following claims.

1 253 1 2506 DNA Artificial Synthetic 1 atgaggggga tgctgcccct ctttgagcccaagggccggg tcctcctggt ggacggccac 60 cacctggcct accgcacctt ccacgccctgaagggcctca ccaccagccg gggggagccg 120 gtgcaggcgg tctacggctt cgccaagagcctcctcaagg ccctcaagga ggacggggac 180 gcggtgatcg tggtctttga cgccaaggccccctccttcc gccacgaggc ctacgggggg 240 tacaaggcgg gccgggcccc cacgccggaggactttcccc ggcaactcgc cctcatcaag 300 gagctggtgg acctcctggg gctggcgcgcctcgaggtcc cgggctacga ggcggacgac 360 gtcctggcca gcctggccaa gaaggcggaaaaggagggct acgaggtccg catcctcacc 420 gccgacaaag acctttacca gctcctttccgaccgcatcc acgtcctcca ccccgagggg 480 tacctcatca ccccggcctg gctttgggaaaagtacggcc tgaggcccga ccagtgggcc 540 gactaccggg ccctgaccgg ggacgagtccgacaaccttc ccggggtcaa gggcatcggg 600 gagaagacgg cgaggaagct tctggaggagtgggggagcc tggaagccct cctcaagaac 660 ctggaccggc tgaagcccgc catccgggagaagatcctgg cccacatgga cgatctgaag 720 ctctcctggg acctggccaa ggtgcgcaccgacctgcccc tggaggtgga cttcgccaaa 780 aggcgggagc ccgaccggga gaggcttagggcctttctgg agaggcttga gtttggcagc 840 ctcctccacg agttcggcct tctggaaagccccaaggccc tggaggaggc cccctggccc 900 ccgccggaag gggccttcgt gggctttgtgctttcccgca aggagcccat gtgggccgat 960 cttctggccc tggccgccgc cagggggggccgggtccacc gggcccccga gccttataaa 1020 gccctcaggg acctgaagga ggcgcgggggcttctcgcca aagacctgag cgttctggcc 1080 ctgagggaag gccttggcct cccgcccggcgacgacccca tgctcctcgc ctacctcctg 1140 gacccttcca acaccacccc cgagggggtggcccggcgct acggcgggga gtggacggag 1200 gaggcggggg agcgggccgc cctttccgagaggctcttcg ccaacctgtg ggggaggctt 1260 gagggggagg agaggctcct ttggctttaccgggaggtgg agaggcccct ttccgctgtc 1320 ctggcccaca tggaggccac gggggtgcgcctggacgtgg cctatctcag ggccttgtcc 1380 ctggaggtgg ccgaggagat cgcccgcctcgaggccgagg tcttccgcct ggccggccac 1440 cccttcaacc tcaactcccg ggaccagctggaaagggtcc tctttgacga gctagggctt 1500 cccgccatcg gcaagacgga gaagaccggcaagcgctcca ccagcgccgc cgtcctggag 1560 gccctccgcg aggcccaccc catcgtggagaagatcctgc agtaccggga gctcaccaag 1620 ctgaagagca cctacattga ccccttgccggacctcatcc accccaggac gggccgcctc 1680 cacacccgct tcaaccagac ggccacggccacgggcaggc taagtagctc cgatcccaac 1740 ctccagaaca tccccgtccg caccccgcttgggcagagga tccgccgggc cttcatcgcc 1800 gaggaggggt ggctattggt ggccctggactatagccaga tagagctcag ggtgctggcc 1860 cacctctccg gcgacgagaa cctgatccgggtcttccagg aggggcggga catccacacg 1920 gagaccgcca gctggatgtt cggcgtcccccgggaggccg tggaccccct gatgcgccgg 1980 gcggccaaga ccatcaactt cggggtcctctacggcatgt cggcccaccg cctctcccag 2040 gagctagcca tcccttacga ggaggcccaggccttcattg agcgctactt tcagagcttc 2100 cccaaggtgc gggcctggat tgagaagaccctggaggagg gcaggaggcg ggggtacgtg 2160 gagaccctct tcggccgccg ccgctacgtgccagacctag aggcccgggt gaagagcgtg 2220 cgggaggcgg ccgagcgcat ggccttcaacatgcccgtcc agggcaccgc cgccgacctc 2280 atgaagctgg ctatggtgaa gctcttccccaggctggagg aaatgggggc caggatgctc 2340 cttcaggtcc acgacgagct ggtcctcgaggccccaaaag agagggcgga ggccgtggcc 2400 cggctggcca aggaggtcat ggagggggtgtatcccctgg ccgtgcccct ggaggtggag 2460 gtggggatag gggaggactg gctctccgccaaggagtgat accacc 2506 2 2496 DNA Artificial Synthetic 2 atggcgatgcttcccctctt tgagcccaaa ggccgcgtgc tcctggtgga cggccaccac 60 ctggcctaccgcaccttctt tgccctcaag ggcctcacca ccagccgcgg cgaacccgtt 120 caggcggtctacggcttcgc caaaagcctc ctcaaggccc tgaaggagga cggggacgtg 180 gtggtggtggtctttgacgc caaggccccc tccttccgcc acgaggccta cgaggcctac 240 aaggcgggccgggcccccac cccggaggac tttccccggc agctggccct catcaaggag 300 ttggtggacctcctaggcct tgtgcggctg gaggttcccg gctttgaggc ggacgacgtg 360 ctggccaccctggccaagcg ggcggaaaag gaggggtacg aggtgcgcat cctcactgcc 420 gaccgcgacctctaccagct cctttcggag cgcatcgcca tcctccaccc tgaggggtac 480 ctgatcaccccggcgtggct ttacgagaag tacggcctgc gcccggagca gtgggtggac 540 taccgggccctggcggggga cccctcggat aacatccccg gggtgaaggg catcggggag 600 aagaccgcccagaggctcat ccgcgagtgg gggagcctgg aaaacctctt ccagcacctg 660 gaccaggtgaagccctcctt gcgggagaag ctccaggcgg gcatggaggc cctggccctt 720 tcccggaagctttcccaggt gcacactgac ctgcccctgg aggtggactt cgggaggcgc 780 cgcacacccaacctggaggg tctgcgggct tttttggagc ggttggagtt tggaagcctc 840 ctccacgagttcggcctcct ggaggggccg aaggcggcag aggaggcccc ctggccccct 900 ccggaaggggcttttttggg cttttccttt tcccgtcccg agcccatgtg ggccgagctt 960 ctggccctggctggggcgtg ggaggggcgc ctccatcggg cacaagaccc ccttaggggc 1020 ctgagggaccttaagggggt gcggggaatc ctggccaagg acctggcggt tttggccctg 1080 cgggagggcctggacctctt cccagaggac gaccccatgc tcctggccta ccttctggac 1140 ccctccaacaccacccctga gggggtggcc cggcgttacg ggggggagtg gacggaggat 1200 gcgggggagagggccctcct ggccgagcgc ctcttccaga ccctaaagga gcgccttaag 1260 ggagaagaacgcctgctttg gctttacgag gaggtggaga agccgctttc ccgggtgttg 1320 gcccggatggaggccacggg ggtccggctg gacgtggcct acctccaggc cctctccctg 1380 gaggtggaggcggaggtgcg ccagctggag gaggaggtct tccgcctggc cggccacccc 1440 ttcaacctcaactcccgcga ccagctggag cgggtgctct ttgacgagct gggcctgcct 1500 gccatcggcaagacggagaa gacggggaaa cgctccacca gcgctgccgt gctggaggcc 1560 ctgcgagaggcccaccccat cgtggaccgc atcctgcagt accgggagct caccaagctc 1620 aagaacacctacatagaccc cctgcccgcc ctggtccacc ccaagaccgg ccggctccac 1680 acccgcttcaaccagacggc caccgccacg ggcaggcttt ccagctccga ccccaacctg 1740 cagaacatccccgtgcgcac ccctctgggc cagcgcatcc gccgagcctt cgtggccgag 1800 gagggctgggtgctggtggt cttggactac agccagattg agcttcgggt cctggcccac 1860 ctctccggggacgagaacct gatccgggtc tttcaggagg ggagggacat ccacacccag 1920 accgccagctggatgttcgg cgtttccccc gaaggggtag accctctgat gcgccgggcg 1980 gccaagaccatcaacttcgg ggtgctctac ggcatgtccg cccaccgcct ctccggggag 2040 ctttccatcccctacgagga ggcggtggcc ttcattgagc gctacttcca gagctacccc 2100 aaggtgcgggcctggattga ggggaccctc gaggagggcc gccggcgggg gtatgtggag 2160 accctcttcggccgccggcg ctatgtgccc gacctcaacg cccgggtgaa gagcgtgcgc 2220 gaggcggcggagcgcatggc cttcaacatg ccggtccagg gcaccgccgc cgacctcatg 2280 aagctggccatggtgcggct tttcccccgg cttcaggaac tgggggcgag gatgcttttg 2340 caggtgcacgacgagctggt cctcgaggcc cccaaggacc gggcggagag ggtagccgct 2400 ttggccaaggaggtcatgga gggggtctgg cccctgcagg tgcccctgga ggtggaggtg 2460 ggcctgggggaggactggct ctccgccaag gagtag 2496 3 2504 DNA Artificial Synthetic 3atggaggcga tgcttccgct ctttgaaccc aaaggccggg tcctcctggt ggacggccac 60cacctggcct accgcacctt cttcgccctg aagggcctca ccacgagccg gggcgaaccg 120gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctgaagga ggacgggtac 180aaggccgtct tcgtggtctt tgacgccaag gccccctcct tccgccacga ggcctacgag 240gcctacaagg cggggagggc cccgaccccc gaggacttcc cccggcagct cgccctcatc 300aaggagctgg tggacctcct ggggtttacc cgcctcgagg tccccggcta cgaggcggac 360gacgttctcg ccaccctggc caagaaggcg gaaaaggagg ggtacgaggt gcgcatcctc 420accgccgacc gcgacctcta ccaactcgtc tccgaccgcg tcgccgtcct ccaccccgag 480ggccacctca tcaccccgga gtggctttgg gagaagtacg gcctcaggcc ggagcagtgg 540gtggacttcc gcgccctcgt gggggacccc tccgacaacc tccccggggt caagggcatc 600ggggagaaga ccgccctcaa gctcctcaag gagtggggaa gcctggaaaa cctcctcaag 660aacctggacc gggtaaagcc agaaaacgtc cgggagaaga tcaaggccca cctggaagac 720ctcaggctct ccttggagct ctcccgggtg cgcaccgacc tccccctgga ggtggacctc 780gcccaggggc gggagcccga ccgggagggg cttagggcct tcctggagag gctggagttc 840ggcagcctcc tccacgagtt cggcctcctg gaggcccccg cccccctgga ggaggccccc 900tggcccccgc cggaaggggc cttcgtgggc ttcgtcctct cccgccccga gcccatgtgg 960gcggagctta aagccctggc cgcctgcagg gacggccggg tgcaccgggc agcagacccc 1020ttggcggggc taaaggacct caaggaggtc cggggcctcc tcgccaagga cctcgccgtc 1080ttggcctcga gggaggggct agacctcgtg cccggggacg accccatgct cctcgcctac 1140ctcctggacc cctccaacac cacccccgag ggggtggcgc ggcgctacgg gggggagtgg 1200acggaggacg ccgcccaccg ggccctcctc tcggagaggc tccatcggaa cctccttaag 1260cgcctcgagg gggaggagaa gctcctttgg ctctaccacg aggtggaaaa gcccctctcc 1320cgggtcctgg cccacatgga ggccaccggg gtacggctgg acgtggccta ccttcaggcc 1380ctttccctgg agcttgcgga ggagatccgc cgcctcgagg aggaggtctt ccgcttggcg 1440ggccacccct tcaacctcaa ctcccgggac cagctggaaa gggtgctctt tgacgagctt 1500aggcttcccg ccttggggaa gacgcaaaag acaggcaagc gctccaccag cgccgcggtg 1560ctggaggccc tacgggaggc ccaccccatc gtggagaaga tcctccagca ccgggagctc 1620accaagctca agaacaccta cgtggacccc ctcccaagcc tcgtccaccc gaggacgggc 1680cgcctccaca cccgcttcaa ccagacggcc acggccacgg ggaggcttag tagctccgac 1740cccaacctgc agaacatccc cgtccgcacc cccttgggcc agaggatccg ccgggccttc 1800gtggccgagg cgggttgggc gttggtggcc ctggactata gccagataga gctccgcgtc 1860ctcgcccacc tctccgggga cgaaaacctg atcagggtct tccaggaggg gaaggacatc 1920cacacccaga ccgcaagctg gatgttcggc gtccccccgg aggccgtgga ccccctgatg 1980cgccgggcgg ccaagacggt gaacttcggc gtcctctacg gcatgtccgc ccataggctc 2040tcccaggagc ttgccatccc ctacgaggag gcggtggcct ttatagaggc tacttccaaa 2100gcttccccaa ggtgcgggcc tggatagaaa agaccctgga ggaggggagg aagcggggct 2160acgtggaaac cctcttcgga agaaggcgct acgtgcccga cctcaacgcc cgggtgaaga 2220gcgtcaggga ggccgcggag cgcatggcct tcaacatgcc cgtccagggc accgccgccg 2280acctcatgaa gctcgccatg gtgaagctct tcccccgcct ccgggagatg ggggcccgca 2340tgctcctcca ggtccacgac gagctcctcc tggaggcccc ccaagcgcgg gccgaggagg 2400tggcggcttt ggccaaggag gccatggaga aggcctatcc cctcgccgtg cccctggagg 2460tggaggtggg gatgggggag gactggcttt ccgccaaggg ttag 2504 4 832 PRTArtificial Synthetic 4 Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys GlyArg Val Leu Leu 1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr PheHis Ala Leu Lys Gly 20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln AlaVal Tyr Gly Phe Ala 35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp GlyAsp Ala Val Ile Val 50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg HisGlu Ala Tyr Gly Gly 65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro GluAsp Phe Pro Arg Gln Leu 85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu LeuGly Leu Ala Arg Leu Glu 100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp ValLeu Ala Ser Leu Ala Lys Lys 115 120 125 Ala Glu Lys Glu Gly Tyr Glu ValArg Ile Leu Thr Ala Asp Lys Asp 130 135 140 Leu Tyr Gln Leu Leu Ser AspArg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr ProAla Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165 170 175 Asp Gln Trp AlaAsp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn 180 185 190 Leu Pro GlyVal Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu 195 200 205 Glu GluTrp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu 210 215 220 LysPro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val 245250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly LeuLeu 275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro ProGlu Gly 290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro MetTrp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly ArgVal His Arg Ala Pro 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu LysGlu Ala Arg Gly Leu Leu 340 345 350 Ala Lys Asp Leu Ser Val Leu Ala LeuArg Glu Gly Leu Gly Leu Pro 355 360 365 Pro Gly Asp Asp Pro Met Leu LeuAla Tyr Leu Leu Asp Pro Ser Asn 370 375 380 Thr Thr Pro Glu Gly Val AlaArg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu ArgAla Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu 405 410 415 Trp Gly Arg LeuGlu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420 425 430 Val Glu ArgPro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly 435 440 445 Val ArgLeu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala 450 455 460 GluGlu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg500 505 510 Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His ProIle 515 520 525 Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu LysSer Thr 530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg ThrGly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala ThrGly Arg Leu Ser Ser 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro ValArg Thr Pro Leu Gly Gln 580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala GluGlu Gly Trp Leu Leu Val Ala 595 600 605 Leu Asp Tyr Ser Gln Ile Glu LeuArg Val Leu Ala His Leu Ser Gly 610 615 620 Asp Glu Asn Leu Ile Arg ValPhe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser TrpMet Phe Gly Val Pro Arg Glu Ala Val Asp Pro 645 650 655 Leu Met Arg ArgAla Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 660 665 670 Met Ser AlaHis Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680 685 Ala GlnAla Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 690 695 700 AlaTrp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg 725730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val LysLeu 755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu GlnVal His 770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala GluAla Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val TyrPro Leu Ala Val Pro 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu AspTrp Leu Ser Ala Lys Glu 820 825 830 5 831 PRT Artificial Synthetic 5 MetAla Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu Val 1 5 10 15Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly Leu 20 25 30Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala Lys 35 40 45Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Val Val Val Val Val 50 55 60Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu Ala Tyr 65 70 7580 Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu Ala 85 9095 Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Val Arg Leu Glu Val 100105 110 Pro Gly Phe Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys Arg Ala115 120 125 Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg AspLeu 130 135 140 Tyr Gln Leu Leu Ser Glu Arg Ile Ala Ile Leu His Pro GluGly Tyr 145 150 155 160 Leu Ile Thr Pro Ala Trp Leu Tyr Glu Lys Tyr GlyLeu Arg Pro Glu 165 170 175 Gln Trp Val Asp Tyr Arg Ala Leu Ala Gly AspPro Ser Asp Asn Ile 180 185 190 Pro Gly Val Lys Gly Ile Gly Glu Lys ThrAla Gln Arg Leu Ile Arg 195 200 205 Glu Trp Gly Ser Leu Glu Asn Leu PheGln His Leu Asp Gln Val Lys 210 215 220 Pro Ser Leu Arg Glu Lys Leu GlnAla Gly Met Glu Ala Leu Ala Leu 225 230 235 240 Ser Arg Lys Leu Ser GlnVal His Thr Asp Leu Pro Leu Glu Val Asp 245 250 255 Phe Gly Arg Arg ArgThr Pro Asn Leu Glu Gly Leu Arg Ala Phe Leu 260 265 270 Glu Arg Leu GluPhe Gly Ser Leu Leu His Glu Phe Gly Leu Leu Glu 275 280 285 Gly Pro LysAla Ala Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly Ala 290 295 300 Phe LeuGly Phe Ser Phe Ser Arg Pro Glu Pro Met Trp Ala Glu Leu 305 310 315 320Leu Ala Leu Ala Gly Ala Trp Glu Gly Arg Leu His Arg Ala Gln Asp 325 330335 Pro Leu Arg Gly Leu Arg Asp Leu Lys Gly Val Arg Gly Ile Leu Ala 340345 350 Lys Asp Leu Ala Val Leu Ala Leu Arg Glu Gly Leu Asp Leu Phe Pro355 360 365 Glu Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser AsnThr 370 375 380 Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp ThrGlu Asp 385 390 395 400 Ala Gly Glu Arg Ala Leu Leu Ala Glu Arg Leu PheGln Thr Leu Lys 405 410 415 Glu Arg Leu Lys Gly Glu Glu Arg Leu Leu TrpLeu Tyr Glu Glu Val 420 425 430 Glu Lys Pro Leu Ser Arg Val Leu Ala ArgMet Glu Ala Thr Gly Val 435 440 445 Arg Leu Asp Val Ala Tyr Leu Gln AlaLeu Ser Leu Glu Val Glu Ala 450 455 460 Glu Val Arg Gln Leu Glu Glu GluVal Phe Arg Leu Ala Gly His Pro 465 470 475 480 Phe Asn Leu Asn Ser ArgAsp Gln Leu Glu Arg Val Leu Phe Asp Glu 485 490 495 Leu Gly Leu Pro AlaIle Gly Lys Thr Glu Lys Thr Gly Lys Arg Ser 500 505 510 Thr Ser Ala AlaVal Leu Glu Ala Leu Arg Glu Ala His Pro Ile Val 515 520 525 Asp Arg IleLeu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Asn Thr Tyr 530 535 540 Ile AspPro Leu Pro Ala Leu Val His Pro Lys Thr Gly Arg Leu His 545 550 555 560Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser Ser 565 570575 Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln Arg 580585 590 Ile Arg Arg Ala Phe Val Ala Glu Glu Gly Trp Val Leu Val Val Leu595 600 605 Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser GlyAsp 610 615 620 Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile HisThr Gln 625 630 635 640 Thr Ala Ser Trp Met Phe Gly Val Ser Pro Glu GlyVal Asp Pro Leu 645 650 655 Met Arg Arg Ala Ala Lys Thr Ile Asn Phe GlyVal Leu Tyr Gly Met 660 665 670 Ser Ala His Arg Leu Ser Gly Glu Leu SerIle Pro Tyr Glu Glu Ala 675 680 685 Val Ala Phe Ile Glu Arg Tyr Phe GlnSer Tyr Pro Lys Val Arg Ala 690 695 700 Trp Ile Glu Gly Thr Leu Glu GluGly Arg Arg Arg Gly Tyr Val Glu 705 710 715 720 Thr Leu Phe Gly Arg ArgArg Tyr Val Pro Asp Leu Asn Ala Arg Val 725 730 735 Lys Ser Val Arg GluAla Ala Glu Arg Met Ala Phe Asn Met Pro Val 740 745 750 Gln Gly Thr AlaAla Asp Leu Met Lys Leu Ala Met Val Arg Leu Phe 755 760 765 Pro Arg LeuGln Glu Leu Gly Ala Arg Met Leu Leu Gln Val His Asp 770 775 780 Glu LeuVal Leu Glu Ala Pro Lys Asp Arg Ala Glu Arg Val Ala Ala 785 790 795 800Leu Ala Lys Glu Val Met Glu Gly Val Trp Pro Leu Gln Val Pro Leu 805 810815 Glu Val Glu Val Gly Leu Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825830 6 834 PRT Artificial Synthetic 6 Met Glu Ala Met Leu Pro Leu Phe GluPro Lys Gly Arg Val Leu Leu 1 5 10 15 Val Asp Gly His His Leu Ala TyrArg Thr Phe Phe Ala Leu Lys Gly 20 25 30 Leu Thr Thr Ser Arg Gly Glu ProVal Gln Ala Val Tyr Gly Phe Ala 35 40 45 Lys Ser Leu Leu Lys Ala Leu LysGlu Asp Gly Tyr Lys Ala Val Phe 50 55 60 Val Val Phe Asp Ala Lys Ala ProSer Phe Arg His Glu Ala Tyr Glu 65 70 75 80 Ala Tyr Lys Ala Gly Arg AlaPro Thr Pro Glu Asp Phe Pro Arg Gln 85 90 95 Leu Ala Leu Ile Lys Glu LeuVal Asp Leu Leu Gly Phe Thr Arg Leu 100 105 110 Glu Val Pro Gly Tyr GluAla Asp Asp Val Leu Ala Thr Leu Ala Lys 115 120 125 Lys Ala Glu Lys GluGly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg 130 135 140 Asp Leu Tyr GlnLeu Val Ser Asp Arg Val Ala Val Leu His Pro Glu 145 150 155 160 Gly HisLeu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg 165 170 175 ProGlu Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp 180 185 190Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu 195 200205 Leu Lys Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg 210215 220 Val Lys Pro Glu Asn Val Arg Glu Lys Ile Lys Ala His Leu Glu Asp225 230 235 240 Leu Arg Leu Ser Leu Glu Leu Ser Arg Val Arg Thr Asp LeuPro Leu 245 250 255 Glu Val Asp Leu Ala Gln Gly Arg Glu Pro Asp Arg GluGly Leu Arg 260 265 270 Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu LeuHis Glu Phe Gly 275 280 285 Leu Leu Glu Ala Pro Ala Pro Leu Glu Glu AlaPro Trp Pro Pro Pro 290 295 300 Glu Gly Ala Phe Val Gly Phe Val Leu SerArg Pro Glu Pro Met Trp 305 310 315 320 Ala Glu Leu Lys Ala Leu Ala AlaCys Arg Asp Gly Arg Val His Arg 325 330 335 Ala Ala Asp Pro Leu Ala GlyLeu Lys Asp Leu Lys Glu Val Arg Gly 340 345 350 Leu Leu Ala Lys Asp LeuAla Val Leu Ala Ser Arg Glu Gly Leu Asp 355 360 365 Leu Val Pro Gly AspAsp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro 370 375 380 Ser Asn Thr ThrPro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp 385 390 395 400 Thr GluAsp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu His Arg 405 410 415 AsnLeu Leu Lys Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr 420 425 430His Glu Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala 435 440445 Thr Gly Val Arg Leu Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu Glu 450455 460 Leu Ala Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala465 470 475 480 Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu ArgVal Leu 485 490 495 Phe Asp Glu Leu Arg Leu Pro Ala Leu Gly Lys Thr GlnLys Thr Gly 500 505 510 Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala LeuArg Glu Ala His 515 520 525 Pro Ile Val Glu Lys Ile Leu Gln His Arg GluLeu Thr Lys Leu Lys 530 535 540 Asn Thr Tyr Val Asp Pro Leu Pro Ser LeuVal His Pro Arg Thr Gly 545 550 555 560 Arg Leu His Thr Arg Phe Asn GlnThr Ala Thr Ala Thr Gly Arg Leu 565 570 575 Ser Ser Ser Asp Pro Asn LeuGln Asn Ile Pro Val Arg Thr Pro Leu 580 585 590 Gly Gln Arg Ile Arg ArgAla Phe Val Ala Glu Ala Gly Trp Ala Leu 595 600 605 Val Ala Leu Asp TyrSer Gln Ile Glu Leu Arg Val Leu Ala His Leu 610 615 620 Ser Gly Asp GluAsn Leu Ile Arg Val Phe Gln Glu Gly Lys Asp Ile 625 630 635 640 His ThrGln Thr Ala Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val 645 650 655 AspPro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu 660 665 670Tyr Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr 675 680685 Glu Glu Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys 690695 700 Val Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly705 710 715 720 Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro AspLeu Asn 725 730 735 Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg MetAla Phe Asn 740 745 750 Met Pro Val Gln Gly Thr Ala Ala Asp Leu Met LysLeu Ala Met Val 755 760 765 Lys Leu Phe Pro Arg Leu Arg Glu Met Gly AlaArg Met Leu Leu Gln 770 775 780 Val His Asp Glu Leu Leu Leu Glu Ala ProGln Ala Arg Ala Glu Glu 785 790 795 800 Val Ala Ala Leu Ala Lys Glu AlaMet Glu Lys Ala Tyr Pro Leu Ala 805 810 815 Val Pro Leu Glu Val Glu ValGly Met Gly Glu Asp Trp Leu Ser Ala 820 825 830 Lys Gly 7 2502 DNAArtificial Synthetic 7 atgnnggcga tgcttcccct ctttgagccc aaaggccgggtcctcctggt ggacggccac 60 cacctggcct accgcacctt cttcgccctg aagggcctcaccaccagccg gggcgaaccg 120 gtgcaggcgg tctacggctt cgccaagagc ctcctcaaggccctgaagga ggacggggac 180 nnggcggtgn tcgtggtctt tgacgccaag gccccctccttccgccacga ggcctacgag 240 gcctacaagg cgggccgggc ccccaccccg gaggactttccccggcagct cgccctcatc 300 aaggagctgg tggacctcct ggggcttgcg cgcctcgaggtccccggcta cgaggcggac 360 gacgtnctgg ccaccctggc caagaaggcg gaaaaggaggggtacgaggt gcgcatcctc 420 accgccgacc gcgacctcta ccagctcctt tccgaccgcatcgccgtcct ccaccccgag 480 gggtacctca tcaccccggc gtggctttgg gagaagtacggcctgaggcc ggagcagtgg 540 gtggactacc gggccctggc gggggacccc tccgacaacctccccggggt caagggcatc 600 ggggagaaga ccgcccngaa gctcctcnag gagtgggggagcctggaaaa cctcctcaag 660 aacctggacc gggtgaagcc cgccntccgg gagaagatccaggcccacat ggangacctg 720 angctctcct gggagctntc ccaggtgcgc accgacctgcccctggaggt ggacttcgcc 780 aagnggcggg agcccgaccg ggaggggctt agggcctttctggagaggct ggagtttggc 840 agcctcctcc acgagttcgg cctcctggag ggccccaaggccctggagga ggccccctgg 900 cccccgccgg aaggggcctt cgtgggcttt gtcctttcccgccccgagcc catgtgggcc 960 gagcttctgg ccctggccgc cgccagggag ggccgggtccaccgggcacc agaccccttt 1020 angggcctna gggacctnaa ggaggtgcgg ggnctcctcgccaaggacct ggccgttttg 1080 gccctgaggg agggcctnga cctcntgccc ggggacgaccccatgctcct cgcctacctc 1140 ctggacccct ccaacaccac ccccgagggg gtggcccggcgctacggggg ggagtggacg 1200 gaggangcgg gggagcgggc cctcctntcc gagaggctcttccngaacct nnngcagcgc 1260 cttgaggggg aggagaggct cctttggctt taccaggaggtggagaagcc cctttcccgg 1320 gtcctggccc acatggaggc cacgggggtn cggctggacgtggcctacct ccaggccctn 1380 tccctggagg tggcggagga gatccgccgc ctcgaggaggaggtcttccg cctggccggc 1440 caccccttca acctcaactc ccgggaccag ctggaaagggtgctctttga cgagctnggg 1500 cttcccgcca tcggcaagac ggagaagacn ggcaagcgctccaccagcgc cgccgtgctg 1560 gaggccctnc gngaggccca ccccatcgtg gagaagatcctgcagtaccg ggagctcacc 1620 aagctcaaga acacctacat ngaccccctg ccngncctcgtccaccccag gacgggccgc 1680 ctccacaccc gcttcaacca gacggccacg gccacgggcaggcttagtag ctccgacccc 1740 aacctgcaga acatccccgt ccgcaccccn ctgggccagaggatccgccg ggccttcgtg 1800 gccgaggagg gntgggtgtt ggtggccctg gactatagccagatagagct ccgggtcctg 1860 gcccacctct ccggggacga gaacctgatc cgggtcttccaggaggggag ggacatccac 1920 acccagaccg ccagctggat gttcggcgtc cccccggaggccgtggaccc cctgatgcgc 1980 cgggcggcca agaccatcaa cttcggggtc ctctacggcatgtccgccca ccgcctctcc 2040 caggagcttg ccatccccta cgaggaggcg gtggccttcattgagcgcta cttccagagc 2100 ttccccaagg tgcgggcctg gattgagaag accctggaggagggcaggag gcgggggtac 2160 gtggagaccc tcttcggccg ccggcgctac gtgcccgacctcaacgcccg ggtgaagagc 2220 gtgcgggagg cggcggagcg catggccttc aacatgcccgtccagggcac cgccgccgac 2280 ctcatgaagc tggccatggt gaagctcttc ccccggctncaggaaatggg ggccaggatg 2340 ctcctncagg tccacgacga gctggtcctc gaggcccccaaagagcgggc ggaggnggtg 2400 gccgctttgg ccaaggaggt catggagggg gtctatcccctggccgtgcc cctggaggtg 2460 gaggtgggga tgggggagga ctggctctcc gccaaggagtag 2502 8 833 PRT Artificial Synthetic 8 Met Xaa Ala Met Leu Pro Leu PheGlu Pro Lys Gly Arg Val Leu Leu 1 5 10 15 Val Asp Gly His His Leu AlaTyr Arg Thr Phe Phe Ala Leu Lys Gly 20 25 30 Leu Thr Thr Ser Arg Gly GluPro Val Gln Ala Val Tyr Gly Phe Ala 35 40 45 Lys Ser Leu Leu Lys Ala LeuLys Glu Asp Gly Asp Ala Val Xaa Val 50 55 60 Val Phe Asp Ala Lys Ala ProSer Phe Arg His Glu Ala Tyr Glu Ala 65 70 75 80 Tyr Lys Ala Gly Arg AlaPro Thr Pro Glu Asp Phe Pro Arg Gln Leu 85 90 95 Ala Leu Ile Lys Glu LeuVal Asp Leu Leu Gly Leu Xaa Arg Leu Glu 100 105 110 Val Pro Gly Tyr GluAla Asp Asp Val Leu Ala Thr Leu Ala Lys Lys 115 120 125 Ala Glu Lys GluGly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg Asp 130 135 140 Leu Tyr GlnLeu Leu Ser Asp Arg Ile Ala Val Leu His Pro Glu Gly 145 150 155 160 TyrLeu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165 170 175Glu Gln Trp Val Asp Tyr Arg Ala Leu Xaa Gly Asp Pro Ser Asp Asn 180 185190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Xaa Lys Leu Leu 195200 205 Xaa Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg Val210 215 220 Lys Pro Xaa Xaa Arg Glu Lys Ile Xaa Ala His Met Glu Asp LeuXaa 225 230 235 240 Leu Ser Xaa Xaa Leu Ser Xaa Val Arg Thr Asp Leu ProLeu Glu Val 245 250 255 Asp Phe Ala Xaa Arg Arg Glu Pro Asp Arg Glu GlyLeu Arg Ala Phe 260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu HisGlu Phe Gly Leu Leu 275 280 285 Glu Xaa Pro Lys Ala Leu Glu Glu Ala ProTrp Pro Pro Pro Glu Gly 290 295 300 Ala Phe Val Gly Phe Val Leu Ser ArgPro Glu Pro Met Trp Ala Glu 305 310 315 320 Leu Leu Ala Leu Ala Ala AlaArg Xaa Gly Arg Val His Arg Ala Xaa 325 330 335 Asp Pro Leu Xaa Gly LeuArg Asp Leu Lys Glu Val Arg Gly Leu Leu 340 345 350 Ala Lys Asp Leu AlaVal Leu Ala Leu Arg Glu Gly Leu Asp Leu Xaa 355 360 365 Pro Gly Asp AspPro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375 380 Thr Thr ProGlu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 AspAla Gly Glu Arg Ala Leu Leu Ser Glu Arg Leu Phe Xaa Asn Leu 405 410 415Xaa Xaa Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Xaa Glu 420 425430 Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala Thr Gly 435440 445 Val Arg Leu Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu Glu Val Ala450 455 460 Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala GlyHis 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg ValLeu Phe Asp 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu LysThr Gly Lys Arg 500 505 510 Ser Thr Ser Ala Ala Val Leu Glu Ala Leu ArgGlu Ala His Pro Ile 515 520 525 Val Glu Lys Ile Leu Gln Tyr Arg Glu LeuThr Lys Leu Lys Asn Thr 530 535 540 Tyr Ile Asp Pro Leu Pro Xaa Leu ValHis Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln ThrAla Thr Ala Thr Gly Arg Leu Ser Ser 565 570 575 Ser Asp Pro Asn Leu GlnAsn Ile Pro Val Arg Thr Pro Leu Gly Gln 580 585 590 Arg Ile Arg Arg AlaPhe Val Ala Glu Glu Gly Trp Xaa Leu Val Ala 595 600 605 Leu Asp Tyr SerGln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly 610 615 620 Asp Glu AsnLeu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 GlnThr Ala Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val Asp Pro 645 650 655Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 660 665670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675680 685 Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly TyrVal 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp LeuAsn Ala Arg 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met AlaPhe Asn Met Pro 740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys LeuAla Met Val Lys Leu 755 760 765 Phe Pro Arg Leu Xaa Glu Met Gly Ala ArgMet Leu Leu Gln Val His 770 775 780 Asp Glu Leu Val Leu Glu Ala Pro LysXaa Arg Ala Glu Xaa Val Ala 785 790 795 800 Ala Leu Ala Lys Glu Val MetGlu Gly Val Tyr Pro Leu Ala Val Pro 805 810 815 Leu Glu Val Glu Val GlyXaa Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825 830 Xaa 9 1647 DNAArtificial Synthetic 9 atgaattcgg ggatgctgcc cctctttgag cccaagggccgggtcctcct ggtggacggc 60 caccacctgg cctaccgcac cttccacgcc ctgaagggcctcaccaccag ccggggggag 120 ccggtgcagg cggtctacgg cttcgccaag agcctcctcaaggccctcaa ggaggacggg 180 gacgcggtga tcgtggtctt tgacgccaag gccccctccttccgccacga ggcctacggg 240 gggtacaagg cgggccgggc ccccacgccg gaggactttccccggcaact cgccctcatc 300 aaggagctgg tggacctcct ggggctggcg cgcctcgaggtcccgggcta cgaggcggac 360 gacgtcctgg ccagcctggc caagaaggcg gaaaaggagggctacgaggt ccgcatcctc 420 accgccgaca aagaccttta ccagctcctt tccgaccgcatccacgtcct ccaccccgag 480 gggtacctca tcaccccggc ctggctttgg gaaaagtacggcctgaggcc cgaccagtgg 540 gccgactacc gggccctgac cggggacgag tccgacaaccttcccggggt caagggcatc 600 ggggagaaga cggcgaggaa gcttctggag gagtgggggagcctggaagc cctcctcaag 660 aacctggacc ggctgaagcc cgccatccgg gagaagatcctggcccacat ggacgatctg 720 aagctctcct gggacctggc caaggtgcgc accgacctgcccctggaggt ggacttcgcc 780 aaaaggcggg agcccgaccg ggagaggctt agggcctttctggagaggct tgagtttggc 840 agcctcctcc acgagttcgg ccttctggaa agccccaaggccctggagga ggccccctgg 900 cccccgccgg aaggggcctt cgtgggcttt gtgctttcccgcaaggagcc catgtgggcc 960 gatcttctgg ccctggccgc cgccaggggg ggccgggtccaccgggcccc cgagccttat 1020 aaagccctca gggacctgaa ggaggcgcgg gggcttctcgccaaagacct gagcgttctg 1080 gccctgaggg aaggccttgg cctcccgccc ggcgacgaccccatgctcct cgcctacctc 1140 ctggaccctt ccaacaccac ccccgagggg gtggcccggcgctacggcgg ggagtggacg 1200 gaggaggcgg gggagcgggc cgccctttcc gagaggctcttcgccaacct gtgggggagg 1260 cttgaggggg aggagaggct cctttggctt taccgggaggtggagaggcc cctttccgct 1320 gtcctggccc acatggaggc cacgggggtg cgcctggacgtggcctatct cagggccttg 1380 tccctggagg tggccgggga gatcgcccgc ctcgaggccgaggtcttccg cctggccggc 1440 caccccttca acctcaactc ccgggaccag ctggaaagggtcctctttga cgagctaggg 1500 cttcccgcca tcggcaagac ggagaagacc ggcaagcgctccaccagcgc cgccgtcctg 1560 gaggccctcc gcgaggccca ccccatcgtg gagaagatcctgcaggcatg caagcttggc 1620 actggccgtc gttttacaac gtcgtga 1647 10 2088DNA Artificial Synthetic 10 atgaattcgg ggatgctgcc cctctttgag cccaagggccgggtcctcct ggtggacggc 60 caccacctgg cctaccgcac cttccacgcc ctgaagggcctcaccaccag ccggggggag 120 ccggtgcagg cggtctacgg cttcgccaag agcctcctcaaggccctcaa ggaggacggg 180 gacgcggtga tcgtggtctt tgacgccaag gccccctccttccgccacga ggcctacggg 240 gggtacaagg cgggccgggc ccccacgccg gaggactttccccggcaact cgccctcatc 300 aaggagctgg tggacctcct ggggctggcg cgcctcgaggtcccgggcta cgaggcggac 360 gacgtcctgg ccagcctggc caagaaggcg gaaaaggagggctacgaggt ccgcatcctc 420 accgccgaca aagaccttta ccagctcctt tccgaccgcatccacgtcct ccaccccgag 480 gggtacctca tcaccccggc ctggctttgg gaaaagtacggcctgaggcc cgaccagtgg 540 gccgactacc gggccctgac cggggacgag tccgacaaccttcccggggt caagggcatc 600 ggggagaaga cggcgaggaa gcttctggag gagtgggggagcctggaagc cctcctcaag 660 aacctggacc ggctgaagcc cgccatccgg gagaagatcctggcccacat ggacgatctg 720 aagctctcct gggacctggc caaggtgcgc accgacctgcccctggaggt ggacttcgcc 780 aaaaggcggg agcccgaccg ggagaggctt agggcctttctggagaggct tgagtttggc 840 agcctcctcc acgagttcgg ccttctggaa agccccaaggccctggagga ggccccctgg 900 cccccgccgg aaggggcctt cgtgggcttt gtgctttcccgcaaggagcc catgtgggcc 960 gatcttctgg ccctggccgc cgccaggggg ggccgggtccaccgggcccc cgagccttat 1020 aaagccctca gggacctgaa ggaggcgcgg gggcttctcgccaaagacct gagcgttctg 1080 gccctgaggg aaggccttgg cctcccgccc ggcgacgaccccatgctcct cgcctacctc 1140 ctggaccctt ccaacaccac ccccgagggg gtggcccggcgctacggcgg ggagtggacg 1200 gaggaggcgg gggagcgggc cgccctttcc gagaggctcttcgccaacct gtgggggagg 1260 cttgaggggg aggagaggct cctttggctt taccgggaggtggagaggcc cctttccgct 1320 gtcctggccc acatggaggc cacgggggtg cgcctggacgtggcctatct cagggccttg 1380 tccctggagg tggccgggga gatcgcccgc ctcgaggccgaggtcttccg cctggccggc 1440 caccccttca acctcaactc ccgggaccag ctggaaagggtcctctttga cgagctaggg 1500 cttcccgcca tcggcaagac ggagaagacc ggcaagcgctccaccagcgc cgccgtcctg 1560 gaggccctcc gcgaggccca ccccatcgtg gagaagatcctgcagtaccg ggagctcacc 1620 aagctgaaga gcacctacat tgaccccttg ccggacctcatccaccccag gacgggccgc 1680 ctccacaccc gcttcaacca gacggccacg gccacgggcaggctaagtag ctccgatccc 1740 aacctccaga acatccccgt ccgcaccccg cttgggcagaggatccgccg ggccttcatc 1800 gccgaggagg ggtggctatt ggtggccctg gactatagccagatagagct cagggtgctg 1860 gcccacctct ccggcgacga gaacctgatc cgggtcttccaggaggggcg ggacatccac 1920 acggagaccg ccagctggat gttcggcgtc ccccgggaggccgtggaccc cctgatgcgc 1980 cgggcggcca agaccatcaa cttcggggtc ctctacggcatgtcggccca ccgcctctcc 2040 caggagctag ctagccatcc cttacgagga ggcccaggccttcattga 2088 11 962 DNA Artificial Synthetic 11 atgaattcgg ggatgctgcccctctttgag cccaagggcc gggtcctcct ggtggacggc 60 caccacctgg cctaccgcaccttccacgcc ctgaagggcc tcaccaccag ccggggggag 120 ccggtgcagg cggtctacggcttcgccaag agcctcctca aggccctcaa ggaggacggg 180 gacgcggtga tcgtggtctttgacgccaag gccccctcct tccgccacga ggcctacggg 240 gggtacaagg cgggccgggcccccacgccg gaggactttc cccggcaact cgccctcatc 300 aaggagctgg tggacctcctggggctggcg cgcctcgagg tcccgggcta cgaggcggac 360 gacgtcctgg ccagcctggccaagaaggcg gaaaaggagg gctacgaggt ccgcatcctc 420 accgccgaca aagacctttaccagcttctt tccgaccgca tccacgtcct ccaccccgag 480 gggtacctca tcaccccggcctggctttgg gaaaagtacg gcctgaggcc cgaccagtgg 540 gccgactacc gggccctgaccggggacgag tccgacaacc ttcccggggt caagggcatc 600 ggggagaaga cggcgaggaagcttctggag gagtggggga gcctggaagc cctcctcaag 660 aacctggacc ggctgaagcccgccatccgg gagaagatcc tggcccacat ggacgatctg 720 aagctctcct gggacctggccaaggtgcgc accgacctgc ccctggaggt ggacttcgcc 780 aaaaggcggg agcccgaccgggagaggctt agggcctttc tggagaggct tgagtttggc 840 agcctcctcc acgagttcggccttctggaa agccccaagt catggagggg gtgtatcccc 900 tggccgtgcc cctggaggtggaggtgggga taggggagga ctggctctcc gccaaggagt 960 ga 962 12 1600 DNAArtificial Synthetic 12 atggaattcg gggatgctgc ccctctttga gcccaagggccgggtcctcc tggtggacgg 60 ccaccacctg gcctaccgca ccttccacgc cctgaagggcctcaccacca gccgggggga 120 gccggtgcag gcggtctacg gcttcgccaa gagcctcctcaaggccctca aggaggacgg 180 ggacgcggtg atcgtggtct ttgacgccaa ggccccctccttccgccacg aggcctacgg 240 ggggtacaag gcgggccggg cccccacgcc ggaggactttccccggcaac tcgccctcat 300 caaggagctg gtggacctcc tggggctggc gcgcctcgaggtcccgggct acgaggcgga 360 cgacgtcctg gccagcctgg ccaagaaggc ggaaaaggagggctacgagg tccgcatcct 420 caccgccgac aaagaccttt accagctcct ttccgaccgcatccacgtcc tccaccccga 480 ggggtacctc atcaccccgg cctggctttg ggaaaagtacggcctgaggc ccgaccagtg 540 ggccgactac cgggccctga ccggggacga gtccgacaaccttcccgggg tcaagggcat 600 cggggagaag acggcgagga agcttctgga ggagtgggggagcctggaag ccctcctcaa 660 gaacctggac cggctgaagc ccgccatccg ggagaagatcctggcccaca tggacgatct 720 gaagctctcc tgggacctgg ccaaggtgcg caccgacctgcccctggagg tggacttcgc 780 caaaaggcgg gagcccgacc gggagaggct tagggcctttctggagaggc ttgagtttgg 840 cagcctcctc cacgagttcg gccttctgga aagccccaagatccgccggg ccttcatcgc 900 cgaggagggg tggctattgg tggccctgga ctatagccagatagagctca gggtgctggc 960 ccacctctcc ggcgacgaga acctgatccg ggtcttccaggaggggcggg acatccacac 1020 ggagaccgcc agctggatgt tcggcgtccc ccgggaggccgtggaccccc tgatgcgccg 1080 ggcggccaag accatcaact tcggggtcct ctacggcatgtcggcccacc gcctctccca 1140 ggagctagcc atcccttacg aggaggccca ggccttcattgagcgctact ttcagagctt 1200 ccccaaggtg cgggcctgga ttgagaagac cctggaggagggcaggaggc gggggtacgt 1260 ggagaccctc ttcggccgcc gccgctacgt gccagacctagaggcccggg tgaagagcgt 1320 gcgggaggcg gccgagcgca tggccttcaa catgcccgtccggggcaccg ccgccgacct 1380 catgaagctg gctatggtga agctcttccc caggctggaggaaatggggg ccaggatgct 1440 ccttcaggtc cacgacgagc tggtcctcga ggccccaaaagagagggcgg aggccgtggc 1500 ccggctggcc aaggaggtca tggagggggt gtatcccctggccgtgcccc tggaggtgga 1560 ggtggggata ggggaggact ggctctccgc caaggagtga1600 13 36 DNA Artificial Synthetic 13 cacgaattcg gggatgctgc ccctctttgagcccaa 36 14 34 DNA Artificial Synthetic 14 gtgagatcta tcactccttggcggagagcc agtc 34 15 91 DNA Artificial Synthetic 15 taatacgactcactataggg agaccggaat tcgagctcgc ccgggcgagc tcgaattccg 60 tgtattctatagtgtcacct aaatcgaatt c 91 16 20 DNA Artificial Synthetic 16 taatacgactcactataggg 20 17 27 DNA Artificial Synthetic 17 gaattcgatt taggtgacactatagaa 27 18 31 DNA Artificial Synthetic 18 gtaatcatgg tcatagctggtagcttgcta c 31 19 42 DNA Artificial Synthetic 19 ggatcctcta gagtcgacctgcaggcatgc ctaccttggt ag 42 20 30 DNA Artificial Synthetic 20 ggatcctctagagtcgacct gcaggcatgc 30 21 2502 DNA Artificial Synthetic 21 atgaattcggggatgctgcc cctctttgag cccaagggcc gggtcctcct ggtggacggc 60 caccacctggcctaccgcac cttccacgcc ctgaagggcc tcaccaccag ccggggggag 120 ccggtgcaggcggtctacgg cttcgccaag agcctcctca aggccctcaa ggaggacggg 180 gacgcggtgatcgtggtctt tgacgccaag gccccctcct tccgccacga ggcctacggg 240 gggtacaaggcgggccgggc ccccacgccg gaggactttc cccggcaact cgccctcatc 300 aaggagctggtggacctcct ggggctggcg cgcctcgagg tcccgggcta cgaggcggac 360 gacgtcctggccagcctggc caagaaggcg gaaaaggagg gctacgaggt ccgcatcctc 420 accgccgacaaagaccttta ccagctcctt tccgaccgca tccacgtcct ccaccccgag 480 gggtacctcatcaccccggc ctggctttgg gaaaagtacg gcctgaggcc cgaccagtgg 540 gccgactaccgggccctgac cggggacgag tccgacaacc ttcccggggt caagggcatc 600 ggggagaagacggcgaggaa gcttctggag gagtggggga gcctggaagc cctcctcaag 660 aacctggaccggctgaagcc cgccatccgg gagaagatcc tggcccacat ggacgatctg 720 aagctctcctgggacctggc caaggtgcgc accgacctgc ccctggaggt ggacttcgcc 780 aaaaggcgggagcccgaccg ggagaggctt agggcctttc tggagaggct tgagtttggc 840 agcctcctccacgagttcgg ccttctggaa agccccaagg ccctggagga ggccccctgg 900 cccccgccggaaggggcctt cgtgggcttt gtgctttccc gcaaggagcc catgtgggcc 960 gatcttctggccctggccgc cgccaggggg ggccgggtcc accgggcccc cgagccttat 1020 aaagccctcagggacctgaa ggaggcgcgg gggcttctcg ccaaagacct gagcgttctg 1080 gccctgagggaaggccttgg cctcccgccc ggcgacgacc ccatgctcct cgcctacctc 1140 ctggacccttccaacaccac ccccgagggg gtggcccggc gctacggcgg ggagtggacg 1200 gaggaggcgggggagcgggc cgccctttcc gagaggctct tcgccaacct gtgggggagg 1260 cttgagggggaggagaggct cctttggctt taccgggagg tggagaggcc cctttccgct 1320 gtcctggcccacatggaggc cacgggggtg cgcctggacg tggcctatct cagggccttg 1380 tccctggaggtggccgggga gatcgcccgc ctcgaggccg aggtcttccg cctggccggc 1440 caccccttcaacctcaactc ccgggaccag ctggaaaggg tcctctttga cgagctaggg 1500 cttcccgccatcggcaagac ggagaagacc ggcaagcgct ccaccagcgc cgccgtcctg 1560 gaggccctccgcgaggccca ccccatcgtg gagaagatcc tgcagtaccg ggagctcacc 1620 aagctgaagagcacctacat tgaccccttg ccggacctca tccaccccag gacgggccgc 1680 ctccacacccgcttcaacca gacggccacg gccacgggca ggctaagtag ctccgatccc 1740 aacctccagaacatccccgt ccgcaccccg cttgggcaga ggatccgccg ggccttcatc 1800 gccgaggaggggtggctatt ggtggccctg gactatagcc agatagagct cagggtgctg 1860 gcccacctctccggcgacga gaacctgatc cgggtcttcc aggaggggcg ggacatccac 1920 acggagaccgccagctggat gttcggcgtc ccccgggagg ccgtggaccc cctgatgcgc 1980 cgggcggccaagaccatcaa cttcggggtc ctctacggca tgtcggccca ccgcctctcc 2040 caggagctagccatccctta cgaggaggcc caggccttca ttgagcgcta ctttcagagc 2100 ttccccaaggtgcgggcctg gattgagaag accctggagg agggcaggag gcgggggtac 2160 gtggagaccctcttcggccg ccgccgctac gtgccagacc tagaggcccg ggtgaagagc 2220 gtgcgggaggcggccgagcg catggccttc aacatgcccg tccggggcac cgccgccgac 2280 ctcatgaagctggctatggt gaagctcttc cccaggctgg aggaaatggg ggccaggatg 2340 ctccttcaggtccacgacga gctggtcctc gaggccccaa aagagagggc ggaggccgtg 2400 gcccggctggccaaggaggt catggagggg gtgtatcccc tggccgtgcc cctggaggtg 2460 gaggtggggataggggagga ctggctctcc gccaaggagt ga 2502 22 19 DNA Artificial Synthetic22 gatttaggtg acactatag 19 23 50 DNA Artificial Synthetic 23 acacaggtaccacatggtac aagaggcaag agagacgaca cagcagaaac 50 24 15 PRT ArtificialSynthetic 24 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Ile Asn Ser1 5 10 15 25 969 DNA Artificial Synthetic 25 atggctagca tgactggtggacagcaaatg ggtcggatca attcggggat gctgcccctc 60 tttgagccca agggccgggtcctcctggtg gacggccacc acctggccta ccgcaccttc 120 cacgccctga agggcctcaccaccagccgg ggggagccgg tgcaggcggt ctacggcttc 180 gccaagagcc tcctcaaggccctcaaggag gacggggacg cggtgatcgt ggtctttgac 240 gccaaggccc cctccttccgccacgaggcc tacggggggt acaaggcggg ccgggccccc 300 acgccggagg actttccccggcaactcgcc ctcatcaagg agctggtgga cctcctgggg 360 ctggcgcgcc tcgaggtcccgggctacgag gcggacgacg tcctggccag cctggccaag 420 aaggcggaaa aggagggctacgaggtccgc atcctcaccg ccgacaaaga cctttaccag 480 cttctttccg accgcatccacgtcctccac cccgaggggt acctcatcac cccggcctgg 540 ctttgggaaa agtacggcctgaggcccgac cagtgggccg actaccgggc cctgaccggg 600 gacgagtccg acaaccttcccggggtcaag ggcatcgggg agaagacggc gaggaagctt 660 ctggaggagt gggggagcctggaagccctc ctcaagaacc tggaccggct gaagcccgcc 720 atccgggaga agatcctggcccacatggac gatctgaagc tctcctggga cctggccaag 780 gtgcgcaccg acctgcccctggaggtggac ttcgccaaaa ggcgggagcc cgaccgggag 840 aggcttaggg cctttctggagaggcttgag tttggcagcc tcctccacga gttcggcctt 900 ctggaaagcc ccaagtcatggagggggtgt atcccctggc cgtgcccctg gaggtggagg 960 tggggatag 969 26 948 DNAArtificial Synthetic 26 atggctagca tgactggtgg acagcaaatg ggtcggatcaattcggggat gctgcccctc 60 tttgagccca agggccgggt cctcctggtg gacggccaccacctggccta ccgcaccttc 120 cacgccctga agggcctcac caccagccgg ggggagccggtgcaggcggt ctacggcttc 180 gccaagagcc tcctcaaggc cctcaaggag gacggggacgcggtgatcgt ggtctttgac 240 gccaaggccc cctccttccg ccacgaggcc tacggggggtacaaggcggg ccgggccccc 300 acgccggagg actttccccg gcaactcgcc ctcatcaaggagctggtgga cctcctgggg 360 ctggcgcgcc tcgaggtccc gggctacgag gcggacgacgtcctggccag cctggccaag 420 aaggcggaaa aggagggcta cgaggtccgc atcctcaccgccgacaaaga cctttaccag 480 cttctttccg accgcatcca cgtcctccac cccgaggggtacctcatcac cccggcctgg 540 ctttgggaaa agtacggcct gaggcccgac cagtgggccgactaccgggc cctgaccggg 600 gacgagtccg acaaccttcc cggggtcaag ggcatcggggagaagacggc gaggaagctt 660 ctggaggagt gggggagcct ggaagccctc ctcaagaacctggaccggct gaagcccgcc 720 atccgggaga agatcctggc ccacatggac gatctgaagctctcctggga cctggccaag 780 gtgcgcaccg acctgcccct ggaggtggac ttcgccaaaaggcgggagcc cgaccgggag 840 aggcttaggg cctttctgga gaggcttgag tttggcagcctcctccacga gttcggcctt 900 ctggaaagcc ccaaggccgc actcgagcac caccaccaccaccactga 948 27 206 DNA Artificial Synthetic 27 cgccagggtt ttcccagtcacgacgttgta aaacgacggc cagtgaattg taatacgact 60 cactataggg cgaattcgagctcggtaccc ggggatcctc tagagtcgac ctgcaggcat 120 gcaagcttga gtattctatagtgtcaccta aatagcttgg cgtaatcatg gtcatagctg 180 tttcctgtgt gaaattgttatccgct 206 28 21 DNA Artificial Synthetic 28 aacagctatg accatgatta c 2129 60 DNA Artificial Synthetic 29 gttctctgct ctctggtcgc tgtctcgcttgtgaaacaag cgagacagcg tggtctctcg 60 30 15 DNA Artificial Synthetic 30cgagagacca cgctg 15 31 52 DNA Artificial Synthetic 31 cctttcgctttcttcccttc ctttctcgcc acgttcgccg gctttccccg tc 52 32 26 DNA ArtificialSynthetic 32 agaaaggaag ggaagaaagc gaaagg 26 33 21 DNA ArtificialSynthetic 33 gacggggaaa gccggcgaac g 21 34 20 DNA Artificial Synthetic34 gaaagccggc gaacgtggcg 20 35 21 DNA Artificial Synthetic 35 ggcgaacgtggcgagaaagg a 21 36 42 DNA Artificial Synthetic 36 cctttcgctt tcttcccttcctttctcgcc acgttcgccg gc 42 37 42 DNA Artificial Synthetic 37 cctttcgctctcttcccttc ctttctcgcc acgttcgccg gc 42 38 27 DNA Artificial Synthetic 38agaaaggaag ggaagaaagc gaaaggt 27 39 24 DNA Artificial Synthetic 39gccggcgaac gtggcgagaa agga 24 40 20 DNA Artificial Synthetic 40ggtttttctt tgaggtttag 20 41 19 DNA Artificial Synthetic 41 gcgacactccaccatagat 19 42 19 DNA Artificial Synthetic 42 ctgtcttcac gcagaaagc 1943 19 DNA Artificial Synthetic 43 gcacggtcta cgagacctc 19 44 20 DNAArtificial Synthetic 44 taatacgact cactataggg 20 45 337 RNA ArtificialSynthetic 45 gggaaagcuu gcaugccugc aggucgacuc uagaggaucu acuagucauauggauucugu 60 cuucacgcag aaagcgucug gccauggcgu uaguaugagu gucgugcagccuccaggacc 120 cccccucccg ggagaggcau aguggucugc ggaaccggug aguacaccggaauugccagg 180 acgaccgggu ccuuucuugg auaaacccgc ucaaugccug gagauuugggcgugcccccg 240 caagacugcu agccgaguag uguugggucg cgaaaggccu ugugguacugccugauaggg 300 ugccugcgag ugccccggga ggucucguag accgugc 337 46 19 DNAArtificial Synthetic 46 ccggtcgtcc tggcaatcc 19 47 24 DNA ArtificialSynthetic 47 gtttatccaa gaaaggaccc ggtc 24 48 30 DNA ArtificialSynthetic 48 cagggtgaag ggaagaagaa agcgaaaggt 30 49 30 DNA ArtificialSynthetic 49 cagggggaag ggaagaagaa agcgaaaggt 30 50 22 DNA ArtificialSynthetic 50 ttcttttcac cagcgagacg gg 22 51 22 DNA Artificial Synthetic51 attgggcgcc agggtggttt tt 22 52 53 DNA Artificial Synthetic 52cccgtctcgc tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgc 53 53 31 DNAArtificial Synthetic 53 gaattcgatt taggtgacac tatagaatac a 31 54 42 DNAArtificial Synthetic 54 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gc42 55 24 DNA Artificial Synthetic 55 gccggcgaac gtggcgagaa agga 24 56 26DNA Artificial Synthetic 56 cagaaggaag ggaagaaagc gaaagg 26 57 26 DNAArtificial Synthetic 57 cagggggaag ggaagaaagc gaaagg 26 58 26 DNAArtificial Synthetic 58 cagggtacag ggaagaaagc gaaagg 26 59 42 DNAArtificial Synthetic 59 gggaaagtcc tcggagccgc gcgggacgag cgtgggggcc cg42 60 963 DNA Artificial Synthetic 60 atg gct agc atg act ggt gga cagcaa atg ggt cgg atc aat tcg ggg 48 Met Ala Ser Met Thr Gly Gly Gln GlnMet Gly Arg Ile Asn Ser Gly 1 5 10 15 atg ctg ccc ctc ttt gag ccc aagggc cgg gtc ctc ctg gtg gac ggc 96 Met Leu Pro Leu Phe Glu Pro Lys GlyArg Val Leu Leu Val Asp Gly 20 25 30 cac cac ctg gcc tac cgc acc ttc cacgcc ctg aag ggc ctc acc acc 144 His His Leu Ala Tyr Arg Thr Phe His AlaLeu Lys Gly Leu Thr Thr 35 40 45 agc cgg ggg gag ccg gtg cag gcg gtc tacggc ttc gcc aag agc ctc 192 Ser Arg Gly Glu Pro Val Gln Ala Val Tyr GlyPhe Ala Lys Ser Leu 50 55 60 ctc aag gcc ctc aag gag gac ggg gac gcg gtgatc gtg gtc ttt gac 240 Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val IleVal Val Phe Asp 65 70 75 80 gcc aag gcc ccc tcc ttc cgc cac gag gcc tacggg ggg tac aag gcg 288 Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr GlyGly Tyr Lys Ala 85 90 95 ggc cgg gcc ccc acg ctc gtc ccg cgc ggc tcc gaggac ttt ccc cgg 336 Gly Arg Ala Pro Thr Leu Val Pro Arg Gly Ser Glu AspPhe Pro Arg 100 105 110 caa ctc gcc ctc atc aag gag ctg gtg gac ctc ctgggg ctg gcg cgc 384 Gln Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu GlyLeu Ala Arg 115 120 125 ctc gag gtc ccg ggc tac gag gcg gac gac gtc ctggcc agc ctg gcc 432 Leu Glu Val Pro Gly Tyr Glu Ala Asp Asp Val Leu AlaSer Leu Ala 130 135 140 aag aag gcg gaa aag gag ggc tac gag gtc cgc atcctc acc gcc gac 480 Lys Lys Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile LeuThr Ala Asp 145 150 155 160 aaa gac ctt tac cag ctc ctt tcc gac cgc atccac gtc ctc cac ccc 528 Lys Asp Leu Tyr Gln Leu Leu Ser Asp Arg Ile HisVal Leu His Pro 165 170 175 gag ggg tac ctc atc acc ccg gcc tgg ctt tgggaa aag tac ggc ctg 576 Glu Gly Tyr Leu Ile Thr Pro Ala Trp Leu Trp GluLys Tyr Gly Leu 180 185 190 agg ccc gac cag tgg gcc gac tac cgg gcc ctgacc ggg gac gag tcc 624 Arg Pro Asp Gln Trp Ala Asp Tyr Arg Ala Leu ThrGly Asp Glu Ser 195 200 205 gac aac ctt ccc ggg gtc aag ggc atc ggg gagaag acg gcg agg aag 672 Asp Asn Leu Pro Gly Val Lys Gly Ile Gly Glu LysThr Ala Arg Lys 210 215 220 ctt ctg gag gag tgg ggg agc ctg gaa gcc ctcctc aag aac ctg gac 720 Leu Leu Glu Glu Trp Gly Ser Leu Glu Ala Leu LeuLys Asn Leu Asp 225 230 235 240 cgg ctg aag ccc gcc atc cgg gag aag atcctg gcc cac atg gac gat 768 Arg Leu Lys Pro Ala Ile Arg Glu Lys Ile LeuAla His Met Asp Asp 245 250 255 ctg aag ctc tcc tgg gac ctg gcc aag gtgcgc acc gac ctg ccc ctg 816 Leu Lys Leu Ser Trp Asp Leu Ala Lys Val ArgThr Asp Leu Pro Leu 260 265 270 gag gtg gac ttc gcc aaa agg cgg gag cccgac cgg gag agg ctt agg 864 Glu Val Asp Phe Ala Lys Arg Arg Glu Pro AspArg Glu Arg Leu Arg 275 280 285 gcc ttt ctg gag agg ctt gag ttt ggc agcctc ctc cac gag ttc ggc 912 Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser LeuLeu His Glu Phe Gly 290 295 300 ctt ctg gaa agc ccc aag gcc gca ctc gagcac cac cac cac cac cac 960 Leu Leu Glu Ser Pro Lys Ala Ala Leu Glu HisHis His His His His 305 310 315 320 tga 963 61 320 PRT ArtificialSynthetic 61 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Ile Asn SerGly 1 5 10 15 Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu ValAsp Gly 20 25 30 His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly LeuThr Thr 35 40 45 Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala LysSer Leu 50 55 60 Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val ValPhe Asp 65 70 75 80 Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly GlyTyr Lys Ala 85 90 95 Gly Arg Ala Pro Thr Leu Val Pro Arg Gly Ser Glu AspPhe Pro Arg 100 105 110 Gln Leu Ala Leu Ile Lys Glu Leu Val Asp Leu LeuGly Leu Ala Arg 115 120 125 Leu Glu Val Pro Gly Tyr Glu Ala Asp Asp ValLeu Ala Ser Leu Ala 130 135 140 Lys Lys Ala Glu Lys Glu Gly Tyr Glu ValArg Ile Leu Thr Ala Asp 145 150 155 160 Lys Asp Leu Tyr Gln Leu Leu SerAsp Arg Ile His Val Leu His Pro 165 170 175 Glu Gly Tyr Leu Ile Thr ProAla Trp Leu Trp Glu Lys Tyr Gly Leu 180 185 190 Arg Pro Asp Gln Trp AlaAsp Tyr Arg Ala Leu Thr Gly Asp Glu Ser 195 200 205 Asp Asn Leu Pro GlyVal Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys 210 215 220 Leu Leu Glu GluTrp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp 225 230 235 240 Arg LeuLys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp 245 250 255 LeuLys Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu 260 265 270Glu Val Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg 275 280285 Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly 290295 300 Leu Leu Glu Ser Pro Lys Ala Ala Leu Glu His His His His His His305 310 315 320 62 20 DNA Artificial Synthetic 62 cgatctcctc ggccacctcc20 63 20 DNA Artificial Synthetic 63 ggcggtgccc tggacgggca 20 64 20 DNAArtificial Synthetic 64 ccagctcgtt gtggacctga 20 65 2505 DNA ArtificialSynthetic 65 atg aat tcg ggg atg ctg ccc ctc ttt gag ccc aag ggc cgg gtcctc 48 Met Asn Ser Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu 15 10 15 ctg gtg gac ggc cac cac ctg gcc tac cgc acc ttc cac gcc ctg aag96 Leu Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys 20 2530 ggc ctc acc acc agc cgg ggg gag ccg gtg cag gcg gtc tac ggc ttc 144Gly Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe 35 40 45gcc aag agc ctc ctc aag gcc ctc aag gag gac ggg gac gcg gtg atc 192 AlaLys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile 50 55 60 gtggtc ttt gac gcc aag gcc ccc tcc ttc cgc cac gag gcc tac ggg 240 Val ValPhe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly 65 70 75 80 gggtac aag gcg ggc cgg gcc ccc acg ccg gag gac ttt ccc cgg caa 288 Gly TyrLys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln 85 90 95 ctc gccctc atc aag gag ctg gtg gac ctc ctg ggg ctg gcg cgc ctc 336 Leu Ala LeuIle Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu 100 105 110 gag gtcccg ggc tac gag gcg gac gac gtc ctg gcc agc ctg gcc aag 384 Glu Val ProGly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys 115 120 125 aag gcggaa aag gag ggc tac gag gtc cgc atc ctc acc gcc gac aaa 432 Lys Ala GluLys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys 130 135 140 gac ctttac cag ctc ctt tcc gac cgc atc cac gtc ctc cac ccc gag 480 Asp Leu TyrGln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu 145 150 155 160 gggtac ctc atc acc ccg gcc tgg ctt tgg gaa aag tac ggc ctg agg 528 Gly TyrLeu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg 165 170 175 cccgac cag tgg gcc gac tac cgg gcc ctg acc ggg gac gag tcc gac 576 Pro AspGln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp 180 185 190 aacctt ccc ggg gtc aag ggc atc ggg gag aag acg gcg agg aag ctt 624 Asn LeuPro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu 195 200 205 ctggag gag tgg ggg agc ctg gaa gcc ctc ctc aag aac ctg gac cgg 672 Leu GluGlu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg 210 215 220 ctgaag ccc gcc atc cgg gag aag atc ctg gcc cac atg gac gat ctg 720 Leu LysPro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu 225 230 235 240aag ctc tcc tgg gac ctg gcc aag gtg cgc acc gac ctg ccc ctg gag 768 LysLeu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu 245 250 255gtg gac ttc gcc aaa agg cgg gag ccc gac cgg gag agg ctt agg gcc 816 ValAsp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala 260 265 270ttt ctg gag agg ctt gag ttt ggc agc ctc ctc cac gag ttc ggc ctt 864 PheLeu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu 275 280 285ctg gaa agc ccc aag gcc ctg gag gag gcc ccc tgg ccc ccg ccg gaa 912 LeuGlu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu 290 295 300ggg gcc ttc gtg ggc ttt gtg ctt tcc cgc aag gag ccc atg tgg gcc 960 GlyAla Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala 305 310 315320 gat ctt ctg gcc ctg gcc gcc gcc agg ggg ggc cgg gtc cac cgg gcc 1008Asp Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala 325 330335 ccc gag cct tat aaa gcc ctc agg gac ctg aag gag gcg cgg ggg ctt 1056Pro Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu 340 345350 ctc gcc aaa gac ctg agc gtt ctg gcc ctg agg gaa ggc ctt ggc ctc 1104Leu Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu 355 360365 ccg ccc ggc gac gac ccc atg ctc ctc gcc tac ctc ctg gac cct tcc 1152Pro Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser 370 375380 aac acc acc ccc gag ggg gtg gcc cgg cgc tac ggc ggg gag tgg acg 1200Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr 385 390395 400 gag gag gcg ggg gag cgg gcc gcc ctt tcc gag agg ctc ttc gcc aac1248 Glu Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn 405410 415 ctg tgg ggg agg ctt gag ggg gag gag agg ctc ctt tgg ctt tac cgg1296 Leu Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg 420425 430 gag gtg gag agg ccc ctt tcc gct gtc ctg gcc cac atg gag gcc acg1344 Glu Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr 435440 445 ggg gtg cgc ctg gac gtg gcc tat ctc agg gcc ttg tcc ctg gag gtg1392 Gly Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val 450455 460 gcc gag gag atc gcc cgc ctc gag gcc gag gtc ttc cgc ctg gcc ggc1440 Ala Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly 465470 475 480 cac ccc ttc aac ctc aac tcc cgg gac cag ctg gaa agg gtc ctcttt 1488 His Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe485 490 495 gac gag cta ggg ctt ccc gcc atc ggc aag acg gag aag acc ggcaag 1536 Asp Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys500 505 510 cgc tcc acc agc gcc gcc gtc ctg gag gcc ctc cgc gag gcc cacccc 1584 Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro515 520 525 atc gtg gag aag atc ctg cag tac cgg gag ctc acc aag ctg aagagc 1632 Ile Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser530 535 540 acc tac att gac ccc ttg ccg gac ctc atc cac ccc agg acg ggccgc 1680 Thr Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg545 550 555 560 ctc cac acc cgc ttc aac cag acg gcc acg gcc acg ggc aggcta agt 1728 Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg LeuSer 565 570 575 agc tcc gat ccc aac ctc cag aac atc ccc gtc cgc acc ccgctt ggg 1776 Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro LeuGly 580 585 590 cag agg atc cgc cgg gcc ttc atc gcc gag gag ggg tgg ctattg gtg 1824 Gln Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu LeuVal 595 600 605 gcc ctg gac tat agc cag ata gag ctc agg gtg ctg gcc cacctc tcc 1872 Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His LeuSer 610 615 620 ggc gac gag aac ctg atc cgg gtc ttc cag gag ggg cgg gacatc cac 1920 Gly Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp IleHis 625 630 635 640 acg gag acc gcc agc tgg atg ttc ggc gtc ccc cgg gaggcc gtg gac 1968 Thr Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu AlaVal Asp 645 650 655 ccc ctg atg cgc cgg gcg gcc aag acc atc aac ttc ggggtc ctc tac 2016 Pro Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly ValLeu Tyr 660 665 670 ggc atg tcg gcc cac cgc ctc tcc cag gag cta gcc atccct tac gag 2064 Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile ProTyr Glu 675 680 685 gag gcc cag gcc ttc att gag cgc tac ttt cag agc ttcccc aag gtg 2112 Glu Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe ProLys Val 690 695 700 cgg gcc tgg att gag aag acc ctg gag gag ggc agg aggcgg ggg tac 2160 Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg ArgGly Tyr 705 710 715 720 gtg gag acc ctc ttc ggc cgc cgc cgc tac gtg ccagac cta gag gcc 2208 Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro AspLeu Glu Ala 725 730 735 cgg gtg aag agc gtg cgg gag gcg gcc gag cgc atggcc ttc aac atg 2256 Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met AlaPhe Asn Met 740 745 750 ccc gtc cag ggc acc gcc gcc gac ctc atg aag ctggct atg gtg aag 2304 Pro Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu AlaMet Val Lys 755 760 765 ctc ttc ccc agg ctg gag gaa atg ggg gcc agg atgctc ctt cag gtc 2352 Leu Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met LeuLeu Gln Val 770 775 780 cac aac gag ctg gtc ctc gag gcc cca aaa gag agggcg gag gcc gtg 2400 His Asn Glu Leu Val Leu Glu Ala Pro Lys Glu Arg AlaGlu Ala Val 785 790 795 800 gcc cgg ctg gcc aag gag gtc atg gag ggg gtgtat ccc ctg gcc gtg 2448 Ala Arg Leu Ala Lys Glu Val Met Glu Gly Val TyrPro Leu Ala Val 805 810 815 ccc ctg gag gtg gag gtg ggg ata ggg gag gactgg ctc tcc gcc aag 2496 Pro Leu Glu Val Glu Val Gly Ile Gly Glu Asp TrpLeu Ser Ala Lys 820 825 830 gag tgatag 2505 Glu 66 833 PRT ArtificialSynthetic 66 Met Asn Ser Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg ValLeu 1 5 10 15 Leu Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His AlaLeu Lys 20 25 30 Gly Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val TyrGly Phe 35 40 45 Ala Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp AlaVal Ile 50 55 60 Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu AlaTyr Gly 65 70 75 80 Gly Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp PhePro Arg Gln 85 90 95 Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly LeuAla Arg Leu 100 105 110 Glu Val Pro Gly Tyr Glu Ala Asp Asp Val Leu AlaSer Leu Ala Lys 115 120 125 Lys Ala Glu Lys Glu Gly Tyr Glu Val Arg IleLeu Thr Ala Asp Lys 130 135 140 Asp Leu Tyr Gln Leu Leu Ser Asp Arg IleHis Val Leu His Pro Glu 145 150 155 160 Gly Tyr Leu Ile Thr Pro Ala TrpLeu Trp Glu Lys Tyr Gly Leu Arg 165 170 175 Pro Asp Gln Trp Ala Asp TyrArg Ala Leu Thr Gly Asp Glu Ser Asp 180 185 190 Asn Leu Pro Gly Val LysGly Ile Gly Glu Lys Thr Ala Arg Lys Leu 195 200 205 Leu Glu Glu Trp GlySer Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg 210 215 220 Leu Lys Pro AlaIle Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu 225 230 235 240 Lys LeuSer Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu 245 250 255 ValAsp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala 260 265 270Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu 275 280285 Leu Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu 290295 300 Gly Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala305 310 315 320 Asp Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val HisArg Ala 325 330 335 Pro Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu AlaArg Gly Leu 340 345 350 Leu Ala Lys Asp Leu Ser Val Leu Ala Leu Arg GluGly Leu Gly Leu 355 360 365 Pro Pro Gly Asp Asp Pro Met Leu Leu Ala TyrLeu Leu Asp Pro Ser 370 375 380 Asn Thr Thr Pro Glu Gly Val Ala Arg ArgTyr Gly Gly Glu Trp Thr 385 390 395 400 Glu Glu Ala Gly Glu Arg Ala AlaLeu Ser Glu Arg Leu Phe Ala Asn 405 410 415 Leu Trp Gly Arg Leu Glu GlyGlu Glu Arg Leu Leu Trp Leu Tyr Arg 420 425 430 Glu Val Glu Arg Pro LeuSer Ala Val Leu Ala His Met Glu Ala Thr 435 440 445 Gly Val Arg Leu AspVal Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val 450 455 460 Ala Glu Glu IleAla Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly 465 470 475 480 His ProPhe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe 485 490 495 AspGlu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys 500 505 510Arg Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro 515 520525 Ile Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser 530535 540 Thr Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg545 550 555 560 Leu His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly ArgLeu Ser 565 570 575 Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg ThrPro Leu Gly 580 585 590 Gln Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu GlyTrp Leu Leu Val 595 600 605 Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg ValLeu Ala His Leu Ser 610 615 620 Gly Asp Glu Asn Leu Ile Arg Val Phe GlnGlu Gly Arg Asp Ile His 625 630 635 640 Thr Glu Thr Ala Ser Trp Met PheGly Val Pro Arg Glu Ala Val Asp 645 650 655 Pro Leu Met Arg Arg Ala AlaLys Thr Ile Asn Phe Gly Val Leu Tyr 660 665 670 Gly Met Ser Ala His ArgLeu Ser Gln Glu Leu Ala Ile Pro Tyr Glu 675 680 685 Glu Ala Gln Ala PheIle Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val 690 695 700 Arg Ala Trp IleGlu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr 705 710 715 720 Val GluThr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala 725 730 735 ArgVal Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met 740 745 750Pro Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys 755 760765 Leu Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val 770775 780 His Asn Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val785 790 795 800 Ala Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro LeuAla Val 805 810 815 Pro Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp LeuSer Ala Lys 820 825 830 Glu 67 20 DNA Artificial Synthetic 67 tggctatagrccagggccac 20 68 2505 DNA Artificial Synthetic 68 atg aat tcg ggg atgctg ccc ctc ttt gag ccc aag ggc cgg gtc ctc 48 Met Asn Ser Gly Met LeuPro Leu Phe Glu Pro Lys Gly Arg Val Leu 1 5 10 15 ctg gtg gac ggc caccac ctg gcc tac cgc acc ttc cac gcc ctg aag 96 Leu Val Asp Gly His HisLeu Ala Tyr Arg Thr Phe His Ala Leu Lys 20 25 30 ggc ctc acc acc agc cggggg gag ccg gtg cag gcg gtc tac ggc ttc 144 Gly Leu Thr Thr Ser Arg GlyGlu Pro Val Gln Ala Val Tyr Gly Phe 35 40 45 gcc aag agc ctc ctc aag gccctc aag gag gac ggg gac gcg gtg atc 192 Ala Lys Ser Leu Leu Lys Ala LeuLys Glu Asp Gly Asp Ala Val Ile 50 55 60 gtg gtc ttt gac gcc aag gcc ccctcc ttc cgc cac gag gcc tac ggg 240 Val Val Phe Asp Ala Lys Ala Pro SerPhe Arg His Glu Ala Tyr Gly 65 70 75 80 ggg tac aag gcg ggc cgg gcc cccacg ccg gag gac ttt ccc cgg caa 288 Gly Tyr Lys Ala Gly Arg Ala Pro ThrPro Glu Asp Phe Pro Arg Gln 85 90 95 ctc gcc ctc atc aag gag ctg gtg gacctc ctg ggg ctg gcg cgc ctc 336 Leu Ala Leu Ile Lys Glu Leu Val Asp LeuLeu Gly Leu Ala Arg Leu 100 105 110 gag gtc ccg ggc tac gag gcg gac gacgtc ctg gcc agc ctg gcc aag 384 Glu Val Pro Gly Tyr Glu Ala Asp Asp ValLeu Ala Ser Leu Ala Lys 115 120 125 aag gcg gaa aag gag ggc tac gag gtccgc atc ctc acc gcc gac aaa 432 Lys Ala Glu Lys Glu Gly Tyr Glu Val ArgIle Leu Thr Ala Asp Lys 130 135 140 gac ctt tac cag ctc ctt tcc gac cgcatc cac gtc ctc cac ccc gag 480 Asp Leu Tyr Gln Leu Leu Ser Asp Arg IleHis Val Leu His Pro Glu 145 150 155 160 ggg tac ctc atc acc ccg gcc tggctt tgg gaa aag tac ggc ctg agg 528 Gly Tyr Leu Ile Thr Pro Ala Trp LeuTrp Glu Lys Tyr Gly Leu Arg 165 170 175 ccc gac cag tgg gcc gac tac cgggcc ctg acc ggg gac gag tcc gac 576 Pro Asp Gln Trp Ala Asp Tyr Arg AlaLeu Thr Gly Asp Glu Ser Asp 180 185 190 aac ctt ccc ggg gtc aag ggc atcggg gag aag acg gcg agg aag ctt 624 Asn Leu Pro Gly Val Lys Gly Ile GlyGlu Lys Thr Ala Arg Lys Leu 195 200 205 ctg gag gag tgg ggg agc ctg gaagcc ctc ctc aag aac ctg gac cgg 672 Leu Glu Glu Trp Gly Ser Leu Glu AlaLeu Leu Lys Asn Leu Asp Arg 210 215 220 ctg aag ccc gcc atc cgg gag aagatc ctg gcc cac atg gac gat ctg 720 Leu Lys Pro Ala Ile Arg Glu Lys IleLeu Ala His Met Asp Asp Leu 225 230 235 240 aag ctc tcc tgg gac ctg gccaag gtg cgc acc gac ctg ccc ctg gag 768 Lys Leu Ser Trp Asp Leu Ala LysVal Arg Thr Asp Leu Pro Leu Glu 245 250 255 gtg gac ttc gcc aaa agg cgggag ccc gac cgg gag agg ctt agg gcc 816 Val Asp Phe Ala Lys Arg Arg GluPro Asp Arg Glu Arg Leu Arg Ala 260 265 270 ttt ctg gag agg ctt gag tttggc agc ctc ctc cac gag ttc ggc ctt 864 Phe Leu Glu Arg Leu Glu Phe GlySer Leu Leu His Glu Phe Gly Leu 275 280 285 ctg gaa agc ccc aag gcc ctggag gag gcc ccc tgg ccc ccg ccg gaa 912 Leu Glu Ser Pro Lys Ala Leu GluGlu Ala Pro Trp Pro Pro Pro Glu 290 295 300 ggg gcc ttc gtg ggc ttt gtgctt tcc cgc aag gag ccc atg tgg gcc 960 Gly Ala Phe Val Gly Phe Val LeuSer Arg Lys Glu Pro Met Trp Ala 305 310 315 320 gat ctt ctg gcc ctg gccgcc gcc agg ggg ggc cgg gtc cac cgg gcc 1008 Asp Leu Leu Ala Leu Ala AlaAla Arg Gly Gly Arg Val His Arg Ala 325 330 335 ccc gag cct tat aaa gccctc agg gac ctg aag gag gcg cgg ggg ctt 1056 Pro Glu Pro Tyr Lys Ala LeuArg Asp Leu Lys Glu Ala Arg Gly Leu 340 345 350 ctc gcc aaa gac ctg agcgtt ctg gcc ctg agg gaa ggc ctt ggc ctc 1104 Leu Ala Lys Asp Leu Ser ValLeu Ala Leu Arg Glu Gly Leu Gly Leu 355 360 365 ccg ccc ggc gac gac cccatg ctc ctc gcc tac ctc ctg gac cct tcc 1152 Pro Pro Gly Asp Asp Pro MetLeu Leu Ala Tyr Leu Leu Asp Pro Ser 370 375 380 aac acc acc ccc gag ggggtg gcc cgg cgc tac ggc ggg gag tgg acg 1200 Asn Thr Thr Pro Glu Gly ValAla Arg Arg Tyr Gly Gly Glu Trp Thr 385 390 395 400 gag gag gcg ggg gagcgg gcc gcc ctt tcc gag agg ctc ttc gcc aac 1248 Glu Glu Ala Gly Glu ArgAla Ala Leu Ser Glu Arg Leu Phe Ala Asn 405 410 415 ctg tgg ggg agg cttgag ggg gag gag agg ctc ctt tgg ctt tac cgg 1296 Leu Trp Gly Arg Leu GluGly Glu Glu Arg Leu Leu Trp Leu Tyr Arg 420 425 430 gag gtg gag agg cccctt tcc gct gtc ctg gcc cac atg gag gcc acg 1344 Glu Val Glu Arg Pro LeuSer Ala Val Leu Ala His Met Glu Ala Thr 435 440 445 ggg gtg cgc ctg gacgtg gcc tat ctc agg gcc ttg tcc ctg gag gtg 1392 Gly Val Arg Leu Asp ValAla Tyr Leu Arg Ala Leu Ser Leu Glu Val 450 455 460 gcc ggg gag atc gcccgc ctc gag gcc gag gtc ttc cgc ctg gcc ggc 1440 Ala Gly Glu Ile Ala ArgLeu Glu Ala Glu Val Phe Arg Leu Ala Gly 465 470 475 480 cac ccc ttc aacctc aac tcc cgg gac cag ctg gaa agg gtc ctc ttt 1488 His Pro Phe Asn LeuAsn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe 485 490 495 gac gag cta gggctt ccc gcc atc ggc aag acg gag aag acc ggc aag 1536 Asp Glu Leu Gly LeuPro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys 500 505 510 cgc tcc acc agcgcc gcc gtc ctg gag gcc ctc cgc gag gcc cac ccc 1584 Arg Ser Thr Ser AlaAla Val Leu Glu Ala Leu Arg Glu Ala His Pro 515 520 525 atc gtg gag aagatc ctg cag tac cgg gag ctc acc aag ctg aag agc 1632 Ile Val Glu Lys IleLeu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser 530 535 540 acc tac att gacccc ttg ccg gac ctc atc cac ccc agg acg ggc cgc 1680 Thr Tyr Ile Asp ProLeu Pro Asp Leu Ile His Pro Arg Thr Gly Arg 545 550 555 560 ctc cac acccgc ttc aac cag acg gcc acg gcc acg ggc agg cta agt 1728 Leu His Thr ArgPhe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser 565 570 575 agc tcc gatccc aac ctc cag aac atc ccc gtc cgc acc ccg ctt ggg 1776 Ser Ser Asp ProAsn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly 580 585 590 cag agg atccgc cgg gcc ttc atc gcc gag gag ggg tgg cta ttg gtg 1824 Gln Arg Ile ArgArg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val 595 600 605 gcc ctg gcctat agc cag ata gag ctc agg gtg ctg gcc cac ctc tcc 1872 Ala Leu Ala TyrSer Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser 610 615 620 ggc gac gagaac ctg atc cgg gtc ttc cag gag ggg cgg gac atc cac 1920 Gly Asp Glu AsnLeu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His 625 630 635 640 acg gagacc gcc agc tgg atg ttc ggc gtc ccc cgg gag gcc gtg gac 1968 Thr Glu ThrAla Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp 645 650 655 ccc ctgatg cgc cgg gcg gcc aag acc atc aac ttc ggg gtc ctc tac 2016 Pro Leu MetArg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr 660 665 670 ggc atgtcg gcc cac cgc ctc tcc cag gag cta gcc atc cct tac gag 2064 Gly Met SerAla His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu 675 680 685 gag gcccag gcc ttc att gag cgc tac ttt cag agc ttc ccc aag gtg 2112 Glu Ala GlnAla Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val 690 695 700 cgg gcctgg att gag aag acc ctg gag gag ggc agg agg cgg ggg tac 2160 Arg Ala TrpIle Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr 705 710 715 720 gtggag acc ctc ttc ggc cgc cgc cgc tac gtg cca gac cta gag gcc 2208 Val GluThr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala 725 730 735 cgggtg aag agc gtg cgg gag gcg gcc gag cgc atg gcc ttc aac atg 2256 Arg ValLys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met 740 745 750 cccgtc cag ggc acc gcc gcc gac ctc atg aag ctg gct atg gtg aag 2304 Pro ValGln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys 755 760 765 ctcttc ccc agg ctg gag gaa atg ggg gcc agg atg ctc ctt cag gtc 2352 Leu PhePro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val 770 775 780 cacgac gag ctg gtc ctc gag gcc cca aaa gag agg gcg gag gcc gtg 2400 His AspGlu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val 785 790 795 800gcc cgg ctg gcc aag gag gtc atg gag ggg gtg tat ccc ctg gcc gtg 2448 AlaArg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val 805 810 815ccc ctg gag gtg gag gtg ggg ata ggg gag gac tgg ctc tcc gcc aag 2496 ProLeu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys 820 825 830gag tgatag 2505 Glu 69 833 PRT Artificial Synthetic 69 Met Asn Ser GlyMet Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu 1 5 10 15 Leu Val AspGly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys 20 25 30 Gly Leu ThrThr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe 35 40 45 Ala Lys SerLeu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile 50 55 60 Val Val PheAsp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly 65 70 75 80 Gly TyrLys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln 85 90 95 Leu AlaLeu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu 100 105 110 GluVal Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys 115 120 125Lys Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys 130 135140 Asp Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu 145150 155 160 Gly Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly LeuArg 165 170 175 Pro Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp GluSer Asp 180 185 190 Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr AlaArg Lys Leu 195 200 205 Leu Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu LysAsn Leu Asp Arg 210 215 220 Leu Lys Pro Ala Ile Arg Glu Lys Ile Leu AlaHis Met Asp Asp Leu 225 230 235 240 Lys Leu Ser Trp Asp Leu Ala Lys ValArg Thr Asp Leu Pro Leu Glu 245 250 255 Val Asp Phe Ala Lys Arg Arg GluPro Asp Arg Glu Arg Leu Arg Ala 260 265 270 Phe Leu Glu Arg Leu Glu PheGly Ser Leu Leu His Glu Phe Gly Leu 275 280 285 Leu Glu Ser Pro Lys AlaLeu Glu Glu Ala Pro Trp Pro Pro Pro Glu 290 295 300 Gly Ala Phe Val GlyPhe Val Leu Ser Arg Lys Glu Pro Met Trp Ala 305 310 315 320 Asp Leu LeuAla Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala 325 330 335 Pro GluPro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu 340 345 350 LeuAla Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu 355 360 365Pro Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser 370 375380 Asn Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr 385390 395 400 Glu Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe AlaAsn 405 410 415 Leu Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp LeuTyr Arg 420 425 430 Glu Val Glu Arg Pro Leu Ser Ala Val Leu Ala His MetGlu Ala Thr 435 440 445 Gly Val Arg Leu Asp Val Ala Tyr Leu Arg Ala LeuSer Leu Glu Val 450 455 460 Ala Gly Glu Ile Ala Arg Leu Glu Ala Glu ValPhe Arg Leu Ala Gly 465 470 475 480 His Pro Phe Asn Leu Asn Ser Arg AspGln Leu Glu Arg Val Leu Phe 485 490 495 Asp Glu Leu Gly Leu Pro Ala IleGly Lys Thr Glu Lys Thr Gly Lys 500 505 510 Arg Ser Thr Ser Ala Ala ValLeu Glu Ala Leu Arg Glu Ala His Pro 515 520 525 Ile Val Glu Lys Ile LeuGln Tyr Arg Glu Leu Thr Lys Leu Lys Ser 530 535 540 Thr Tyr Ile Asp ProLeu Pro Asp Leu Ile His Pro Arg Thr Gly Arg 545 550 555 560 Leu His ThrArg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser 565 570 575 Ser SerAsp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly 580 585 590 GlnArg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val 595 600 605Ala Leu Ala Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser 610 615620 Gly Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His 625630 635 640 Thr Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala ValAsp 645 650 655 Pro Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly ValLeu Tyr 660 665 670 Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala IlePro Tyr Glu 675 680 685 Glu Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln SerPhe Pro Lys Val 690 695 700 Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu GlyArg Arg Arg Gly Tyr 705 710 715 720 Val Glu Thr Leu Phe Gly Arg Arg ArgTyr Val Pro Asp Leu Glu Ala 725 730 735 Arg Val Lys Ser Val Arg Glu AlaAla Glu Arg Met Ala Phe Asn Met 740 745 750 Pro Val Gln Gly Thr Ala AlaAsp Leu Met Lys Leu Ala Met Val Lys 755 760 765 Leu Phe Pro Arg Leu GluGlu Met Gly Ala Arg Met Leu Leu Gln Val 770 775 780 His Asp Glu Leu ValLeu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val 785 790 795 800 Ala Arg LeuAla Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val 805 810 815 Pro LeuGlu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys 820 825 830 Glu70 2505 DNA Artificial Synthetic 70 atg aat tcg ggg atg ctg ccc ctc tttgag ccc aag ggc cgg gtc ctc 48 Met Asn Ser Gly Met Leu Pro Leu Phe GluPro Lys Gly Arg Val Leu 1 5 10 15 ctg gtg gac ggc cac cac ctg gcc taccgc acc ttc cac gcc ctg aag 96 Leu Val Asp Gly His His Leu Ala Tyr ArgThr Phe His Ala Leu Lys 20 25 30 ggc ctc acc acc agc cgg ggg gag ccg gtgcag gcg gtc tac ggc ttc 144 Gly Leu Thr Thr Ser Arg Gly Glu Pro Val GlnAla Val Tyr Gly Phe 35 40 45 gcc aag agc ctc ctc aag gcc ctc aag gag gacggg gac gcg gtg atc 192 Ala Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp GlyAsp Ala Val Ile 50 55 60 gtg gtc ttt gac gcc aag gcc ccc tcc ttc cgc cacgag gcc tac ggg 240 Val Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His GluAla Tyr Gly 65 70 75 80 ggg tac aag gcg ggc cgg gcc ccc acg ccg gag gacttt ccc cgg caa 288 Gly Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp PhePro Arg Gln 85 90 95 ctc gcc ctc atc aag gag ctg gtg gac ctc ctg ggg ctggcg cgc ctc 336 Leu Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu AlaArg Leu 100 105 110 gag gtc ccg ggc tac gag gcg gac gac gtc ctg gcc agcctg gcc aag 384 Glu Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser LeuAla Lys 115 120 125 aag gcg gaa aag gag ggc tac gag gtc cgc atc ctc accgcc gac aaa 432 Lys Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr AlaAsp Lys 130 135 140 gac ctt tac cag ctc ctt tcc gac cgc atc cac gtc ctccac ccc gag 480 Asp Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu HisPro Glu 145 150 155 160 ggg tac ctc atc acc ccg gcc tgg ctt tgg gaa aagtac ggc ctg agg 528 Gly Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys TyrGly Leu Arg 165 170 175 ccc gac cag tgg gcc gac tac cgg gcc ctg acc ggggac gag tcc gac 576 Pro Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly AspGlu Ser Asp 180 185 190 aac ctt ccc ggg gtc aag ggc atc ggg gag aag acggcg agg aag ctt 624 Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr AlaArg Lys Leu 195 200 205 ctg gag gag tgg ggg agc ctg gaa gcc ctc ctc aagaac ctg gac cgg 672 Leu Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys AsnLeu Asp Arg 210 215 220 ctg aag ccc gcc atc cgg gag aag atc ctg gcc cacatg gac gat ctg 720 Leu Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His MetAsp Asp Leu 225 230 235 240 aag ctc tcc tgg gac ctg gcc aag gtg cgc accgac ctg ccc ctg gag 768 Lys Leu Ser Trp Asp Leu Ala Lys Val Arg Thr AspLeu Pro Leu Glu 245 250 255 gtg gac ttc gcc aaa agg cgg gag ccc gac cgggag agg ctt agg gcc 816 Val Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg GluArg Leu Arg Ala 260 265 270 ttt ctg gag agg ctt gag ttt ggc agc ctc ctccac gag ttc ggc ctt 864 Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu HisGlu Phe Gly Leu 275 280 285 ctg gaa agc ccc aag gcc ctg gag gag gcc ccctgg ccc ccg ccg gaa 912 Leu Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro TrpPro Pro Pro Glu 290 295 300 ggg gcc ttc gtg ggc ttt gtg ctt tcc cgc aaggag ccc atg tgg gcc 960 Gly Ala Phe Val Gly Phe Val Leu Ser Arg Lys GluPro Met Trp Ala 305 310 315 320 gat ctt ctg gcc ctg gcc gcc gcc agg gggggc cgg gtc cac cgg gcc 1008 Asp Leu Leu Ala Leu Ala Ala Ala Arg Gly GlyArg Val His Arg Ala 325 330 335 ccc gag cct tat aaa gcc ctc agg gac ctgaag gag gcg cgg ggg ctt 1056 Pro Glu Pro Tyr Lys Ala Leu Arg Asp Leu LysGlu Ala Arg Gly Leu 340 345 350 ctc gcc aaa gac ctg agc gtt ctg gcc ctgagg gaa ggc ctt ggc ctc 1104 Leu Ala Lys Asp Leu Ser Val Leu Ala Leu ArgGlu Gly Leu Gly Leu 355 360 365 ccg ccc ggc gac gac ccc atg ctc ctc gcctac ctc ctg gac cct tcc 1152 Pro Pro Gly Asp Asp Pro Met Leu Leu Ala TyrLeu Leu Asp Pro Ser 370 375 380 aac acc acc ccc gag ggg gtg gcc cgg cgctac ggc ggg gag tgg acg 1200 Asn Thr Thr Pro Glu Gly Val Ala Arg Arg TyrGly Gly Glu Trp Thr 385 390 395 400 gag gag gcg ggg gag cgg gcc gcc ctttcc gag agg ctc ttc gcc aac 1248 Glu Glu Ala Gly Glu Arg Ala Ala Leu SerGlu Arg Leu Phe Ala Asn 405 410 415 ctg tgg ggg agg ctt gag ggg gag gagagg ctc ctt tgg ctt tac cgg 1296 Leu Trp Gly Arg Leu Glu Gly Glu Glu ArgLeu Leu Trp Leu Tyr Arg 420 425 430 gag gtg gag agg ccc ctt tcc gct gtcctg gcc cac atg gag gcc acg 1344 Glu Val Glu Arg Pro Leu Ser Ala Val LeuAla His Met Glu Ala Thr 435 440 445 ggg gtg cgc ctg gac gtg gcc tat ctcagg gcc ttg tcc ctg gag gtg 1392 Gly Val Arg Leu Asp Val Ala Tyr Leu ArgAla Leu Ser Leu Glu Val 450 455 460 gcc ggg gag atc gcc cgc ctc gag gccgag gtc ttc cgc ctg gcc ggc 1440 Ala Gly Glu Ile Ala Arg Leu Glu Ala GluVal Phe Arg Leu Ala Gly 465 470 475 480 cac ccc ttc aac ctc aac tcc cgggac cag ctg gaa agg gtc ctc ttt 1488 His Pro Phe Asn Leu Asn Ser Arg AspGln Leu Glu Arg Val Leu Phe 485 490 495 gac gag cta ggg ctt ccc gcc atcggc aag acg gag aag acc ggc aag 1536 Asp Glu Leu Gly Leu Pro Ala Ile GlyLys Thr Glu Lys Thr Gly Lys 500 505 510 cgc tcc acc agc gcc gcc gtc ctggag gcc ctc cgc gag gcc cac ccc 1584 Arg Ser Thr Ser Ala Ala Val Leu GluAla Leu Arg Glu Ala His Pro 515 520 525 atc gtg gag aag atc ctg cag taccgg gag ctc acc aag ctg aag agc 1632 Ile Val Glu Lys Ile Leu Gln Tyr ArgGlu Leu Thr Lys Leu Lys Ser 530 535 540 acc tac att gac ccc ttg ccg gacctc atc cac ccc agg acg ggc cgc 1680 Thr Tyr Ile Asp Pro Leu Pro Asp LeuIle His Pro Arg Thr Gly Arg 545 550 555 560 ctc cac acc cgc ttc aac cagacg gcc acg gcc acg ggc agg cta agt 1728 Leu His Thr Arg Phe Asn Gln ThrAla Thr Ala Thr Gly Arg Leu Ser 565 570 575 agc tcc gat ccc aac ctc cagaac atc ccc gtc cgc acc ccg ctt ggg 1776 Ser Ser Asp Pro Asn Leu Gln AsnIle Pro Val Arg Thr Pro Leu Gly 580 585 590 cag agg atc cgc cgg gcc ttcatc gcc gag gag ggg tgg cta ttg gtg 1824 Gln Arg Ile Arg Arg Ala Phe IleAla Glu Glu Gly Trp Leu Leu Val 595 600 605 gcc ctg gtc tat agc cag atagag ctc agg gtg ctg gcc cac ctc tcc 1872 Ala Leu Val Tyr Ser Gln Ile GluLeu Arg Val Leu Ala His Leu Ser 610 615 620 ggc gac gag aac ctg atc cgggtc ttc cag gag ggg cgg gac atc cac 1920 Gly Asp Glu Asn Leu Ile Arg ValPhe Gln Glu Gly Arg Asp Ile His 625 630 635 640 acg gag acc gcc agc tggatg ttc ggc gtc ccc cgg gag gcc gtg gac 1968 Thr Glu Thr Ala Ser Trp MetPhe Gly Val Pro Arg Glu Ala Val Asp 645 650 655 ccc ctg atg cgc cgg gcggcc aag acc atc aac ttc ggg gtc ctc tac 2016 Pro Leu Met Arg Arg Ala AlaLys Thr Ile Asn Phe Gly Val Leu Tyr 660 665 670 ggc atg tcg gcc cac cgcctc tcc cag gag cta gcc atc cct tac gag 2064 Gly Met Ser Ala His Arg LeuSer Gln Glu Leu Ala Ile Pro Tyr Glu 675 680 685 gag gcc cag gcc ttc attgag cgc tac ttt cag agc ttc ccc aag gtg 2112 Glu Ala Gln Ala Phe Ile GluArg Tyr Phe Gln Ser Phe Pro Lys Val 690 695 700 cgg gcc tgg att gag aagacc ctg gag gag ggc agg agg cgg ggg tac 2160 Arg Ala Trp Ile Glu Lys ThrLeu Glu Glu Gly Arg Arg Arg Gly Tyr 705 710 715 720 gtg gag acc ctc ttcggc cgc cgc cgc tac gtg cca gac cta gag gcc 2208 Val Glu Thr Leu Phe GlyArg Arg Arg Tyr Val Pro Asp Leu Glu Ala 725 730 735 cgg gtg aag agc gtgcgg gag gcg gcc gag cgc atg gcc ttc aac atg 2256 Arg Val Lys Ser Val ArgGlu Ala Ala Glu Arg Met Ala Phe Asn Met 740 745 750 ccc gtc cag ggc accgcc gcc gac ctc atg aag ctg gct atg gtg aag 2304 Pro Val Gln Gly Thr AlaAla Asp Leu Met Lys Leu Ala Met Val Lys 755 760 765 ctc ttc ccc agg ctggag gaa atg ggg gcc agg atg ctc ctt cag gtc 2352 Leu Phe Pro Arg Leu GluGlu Met Gly Ala Arg Met Leu Leu Gln Val 770 775 780 cac gac gag ctg gtcctc gag gcc cca aaa gag agg gcg gag gcc gtg 2400 His Asp Glu Leu Val LeuGlu Ala Pro Lys Glu Arg Ala Glu Ala Val 785 790 795 800 gcc cgg ctg gccaag gag gtc atg gag ggg gtg tat ccc ctg gcc gtg 2448 Ala Arg Leu Ala LysGlu Val Met Glu Gly Val Tyr Pro Leu Ala Val 805 810 815 ccc ctg gag gtggag gtg ggg ata ggg gag gac tgg ctc tcc gcc aag 2496 Pro Leu Glu Val GluVal Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys 820 825 830 gag tgatag 2505Glu 71 833 PRT Artificial Synthetic 71 Met Asn Ser Gly Met Leu Pro LeuPhe Glu Pro Lys Gly Arg Val Leu 1 5 10 15 Leu Val Asp Gly His His LeuAla Tyr Arg Thr Phe His Ala Leu Lys 20 25 30 Gly Leu Thr Thr Ser Arg GlyGlu Pro Val Gln Ala Val Tyr Gly Phe 35 40 45 Ala Lys Ser Leu Leu Lys AlaLeu Lys Glu Asp Gly Asp Ala Val Ile 50 55 60 Val Val Phe Asp Ala Lys AlaPro Ser Phe Arg His Glu Ala Tyr Gly 65 70 75 80 Gly Tyr Lys Ala Gly ArgAla Pro Thr Pro Glu Asp Phe Pro Arg Gln 85 90 95 Leu Ala Leu Ile Lys GluLeu Val Asp Leu Leu Gly Leu Ala Arg Leu 100 105 110 Glu Val Pro Gly TyrGlu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys 115 120 125 Lys Ala Glu LysGlu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys 130 135 140 Asp Leu TyrGln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu 145 150 155 160 GlyTyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg 165 170 175Pro Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp 180 185190 Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu 195200 205 Leu Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg210 215 220 Leu Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp AspLeu 225 230 235 240 Lys Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp LeuPro Leu Glu 245 250 255 Val Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg GluArg Leu Arg Ala 260 265 270 Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu LeuHis Glu Phe Gly Leu 275 280 285 Leu Glu Ser Pro Lys Ala Leu Glu Glu AlaPro Trp Pro Pro Pro Glu 290 295 300 Gly Ala Phe Val Gly Phe Val Leu SerArg Lys Glu Pro Met Trp Ala 305 310 315 320 Asp Leu Leu Ala Leu Ala AlaAla Arg Gly Gly Arg Val His Arg Ala 325 330 335 Pro Glu Pro Tyr Lys AlaLeu Arg Asp Leu Lys Glu Ala Arg Gly Leu 340 345 350 Leu Ala Lys Asp LeuSer Val Leu Ala Leu Arg Glu Gly Leu Gly Leu 355 360 365 Pro Pro Gly AspAsp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser 370 375 380 Asn Thr ThrPro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr 385 390 395 400 GluGlu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn 405 410 415Leu Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg 420 425430 Glu Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr 435440 445 Gly Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val450 455 460 Ala Gly Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu AlaGly 465 470 475 480 His Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu ArgVal Leu Phe 485 490 495 Asp Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr GluLys Thr Gly Lys 500 505 510 Arg Ser Thr Ser Ala Ala Val Leu Glu Ala LeuArg Glu Ala His Pro 515 520 525 Ile Val Glu Lys Ile Leu Gln Tyr Arg GluLeu Thr Lys Leu Lys Ser 530 535 540 Thr Tyr Ile Asp Pro Leu Pro Asp LeuIle His Pro Arg Thr Gly Arg 545 550 555 560 Leu His Thr Arg Phe Asn GlnThr Ala Thr Ala Thr Gly Arg Leu Ser 565 570 575 Ser Ser Asp Pro Asn LeuGln Asn Ile Pro Val Arg Thr Pro Leu Gly 580 585 590 Gln Arg Ile Arg ArgAla Phe Ile Ala Glu Glu Gly Trp Leu Leu Val 595 600 605 Ala Leu Val TyrSer Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser 610 615 620 Gly Asp GluAsn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His 625 630 635 640 ThrGlu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp 645 650 655Pro Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr 660 665670 Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu 675680 685 Glu Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val690 695 700 Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg GlyTyr 705 710 715 720 Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro AspLeu Glu Ala 725 730 735 Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg MetAla Phe Asn Met 740 745 750 Pro Val Gln Gly Thr Ala Ala Asp Leu Met LysLeu Ala Met Val Lys 755 760 765 Leu Phe Pro Arg Leu Glu Glu Met Gly AlaArg Met Leu Leu Gln Val 770 775 780 His Asp Glu Leu Val Leu Glu Ala ProLys Glu Arg Ala Glu Ala Val 785 790 795 800 Ala Arg Leu Ala Lys Glu ValMet Glu Gly Val Tyr Pro Leu Ala Val 805 810 815 Pro Leu Glu Val Glu ValGly Ile Gly Glu Asp Trp Leu Ser Ala Lys 820 825 830 Glu 72 25 DNAArtificial Synthetic 72 gggataccat gggagtgcag tttgg 25 73 27 DNAArtificial Synthetic 73 ggtaaatttt tctcgtcgac atcccac 27 74 981 DNAArtificial Synthetic 74 atg gga gtg cag ttt ggt gat ttt att cca aaa aatatt atc tcc ttt 48 Met Gly Val Gln Phe Gly Asp Phe Ile Pro Lys Asn IleIle Ser Phe 1 5 10 15 gaa gat tta aaa ggg aaa aaa gta gct att gat ggaatg aat gca tta 96 Glu Asp Leu Lys Gly Lys Lys Val Ala Ile Asp Gly MetAsn Ala Leu 20 25 30 tat cag ttt tta aca tct ata cgt ttg aga gat ggt tctcca ttg aga 144 Tyr Gln Phe Leu Thr Ser Ile Arg Leu Arg Asp Gly Ser ProLeu Arg 35 40 45 aat aga aaa gga gag ata acc tca gca tat aac gga gtt ttttat aaa 192 Asn Arg Lys Gly Glu Ile Thr Ser Ala Tyr Asn Gly Val Phe TyrLys 50 55 60 acc ata cat ttg tta gag aat gat ata act cca atc tgg gtt tttgat 240 Thr Ile His Leu Leu Glu Asn Asp Ile Thr Pro Ile Trp Val Phe Asp65 70 75 80 ggt gag cca cca aag tta aag gag aaa aca agg aaa gtt agg agagag 288 Gly Glu Pro Pro Lys Leu Lys Glu Lys Thr Arg Lys Val Arg Arg Glu85 90 95 atg aaa gag aaa gct gaa ctt aag atg aaa gag gca att aaa aag gag336 Met Lys Glu Lys Ala Glu Leu Lys Met Lys Glu Ala Ile Lys Lys Glu 100105 110 gat ttt gaa gaa gct gct aag tat gca aag agg gtt agc tat cta act384 Asp Phe Glu Glu Ala Ala Lys Tyr Ala Lys Arg Val Ser Tyr Leu Thr 115120 125 ccg aaa atg gtt gaa aac tgc aaa tat ttg tta agt ttg atg ggc att432 Pro Lys Met Val Glu Asn Cys Lys Tyr Leu Leu Ser Leu Met Gly Ile 130135 140 ccg tat gtt gaa gct ccc tct gag gga gag gca caa gca agc tat atg480 Pro Tyr Val Glu Ala Pro Ser Glu Gly Glu Ala Gln Ala Ser Tyr Met 145150 155 160 gca aag aag gga gat gtt tgg gca gtt gta agt caa gat tat gatgcc 528 Ala Lys Lys Gly Asp Val Trp Ala Val Val Ser Gln Asp Tyr Asp Ala165 170 175 ttg tta tat gga gct ccg aga gtt gtt aga aat tta aca act acaaag 576 Leu Leu Tyr Gly Ala Pro Arg Val Val Arg Asn Leu Thr Thr Thr Lys180 185 190 gag atg cca gaa ctt att gaa tta aat gag gtt tta gag gat ttaaga 624 Glu Met Pro Glu Leu Ile Glu Leu Asn Glu Val Leu Glu Asp Leu Arg195 200 205 att tct ttg gat gat ttg ata gat ata gcc ata ttt atg gga actgac 672 Ile Ser Leu Asp Asp Leu Ile Asp Ile Ala Ile Phe Met Gly Thr Asp210 215 220 tat aat cca gga gga gtt aaa gga ata gga ttt aaa agg gct tatgaa 720 Tyr Asn Pro Gly Gly Val Lys Gly Ile Gly Phe Lys Arg Ala Tyr Glu225 230 235 240 ttg gtt aga agt ggt gta gct aag gat gtt ttg aaa aaa gaggtt gaa 768 Leu Val Arg Ser Gly Val Ala Lys Asp Val Leu Lys Lys Glu ValGlu 245 250 255 tac tac gat gag att aag agg ata ttt aaa gag cca aag gttacc gat 816 Tyr Tyr Asp Glu Ile Lys Arg Ile Phe Lys Glu Pro Lys Val ThrAsp 260 265 270 aac tat tca tta agc cta aaa ttg cca gat aaa gag gga attata aaa 864 Asn Tyr Ser Leu Ser Leu Lys Leu Pro Asp Lys Glu Gly Ile IleLys 275 280 285 ttc tta gtt gat gaa aat gac ttt aat tat gat agg gtt aaaaag cat 912 Phe Leu Val Asp Glu Asn Asp Phe Asn Tyr Asp Arg Val Lys LysHis 290 295 300 gtt gat aaa ctc tat aac tta att gca aac aaa act aag caaaaa aca 960 Val Asp Lys Leu Tyr Asn Leu Ile Ala Asn Lys Thr Lys Gln LysThr 305 310 315 320 tta gat gca tgg ttt aaa taa 981 Leu Asp Ala Trp PheLys 325 75 326 PRT Artificial Synthetic 75 Met Gly Val Gln Phe Gly AspPhe Ile Pro Lys Asn Ile Ile Ser Phe 1 5 10 15 Glu Asp Leu Lys Gly LysLys Val Ala Ile Asp Gly Met Asn Ala Leu 20 25 30 Tyr Gln Phe Leu Thr SerIle Arg Leu Arg Asp Gly Ser Pro Leu Arg 35 40 45 Asn Arg Lys Gly Glu IleThr Ser Ala Tyr Asn Gly Val Phe Tyr Lys 50 55 60 Thr Ile His Leu Leu GluAsn Asp Ile Thr Pro Ile Trp Val Phe Asp 65 70 75 80 Gly Glu Pro Pro LysLeu Lys Glu Lys Thr Arg Lys Val Arg Arg Glu 85 90 95 Met Lys Glu Lys AlaGlu Leu Lys Met Lys Glu Ala Ile Lys Lys Glu 100 105 110 Asp Phe Glu GluAla Ala Lys Tyr Ala Lys Arg Val Ser Tyr Leu Thr 115 120 125 Pro Lys MetVal Glu Asn Cys Lys Tyr Leu Leu Ser Leu Met Gly Ile 130 135 140 Pro TyrVal Glu Ala Pro Ser Glu Gly Glu Ala Gln Ala Ser Tyr Met 145 150 155 160Ala Lys Lys Gly Asp Val Trp Ala Val Val Ser Gln Asp Tyr Asp Ala 165 170175 Leu Leu Tyr Gly Ala Pro Arg Val Val Arg Asn Leu Thr Thr Thr Lys 180185 190 Glu Met Pro Glu Leu Ile Glu Leu Asn Glu Val Leu Glu Asp Leu Arg195 200 205 Ile Ser Leu Asp Asp Leu Ile Asp Ile Ala Ile Phe Met Gly ThrAsp 210 215 220 Tyr Asn Pro Gly Gly Val Lys Gly Ile Gly Phe Lys Arg AlaTyr Glu 225 230 235 240 Leu Val Arg Ser Gly Val Ala Lys Asp Val Leu LysLys Glu Val Glu 245 250 255 Tyr Tyr Asp Glu Ile Lys Arg Ile Phe Lys GluPro Lys Val Thr Asp 260 265 270 Asn Tyr Ser Leu Ser Leu Lys Leu Pro AspLys Glu Gly Ile Ile Lys 275 280 285 Phe Leu Val Asp Glu Asn Asp Phe AsnTyr Asp Arg Val Lys Lys His 290 295 300 Val Asp Lys Leu Tyr Asn Leu IleAla Asn Lys Thr Lys Gln Lys Thr 305 310 315 320 Leu Asp Ala Trp Phe Lys325 76 21 DNA Artificial Synthetic 76 gaggtgatac catgggtgtc c 21 77 20DNA Artificial Synthetic 77 gaaactctgc agcgcgtcag 20 78 1023 DNAArtificial Synthetic 78 atg ggt gtc cca att ggt gag att ata cca aga aaagaa att gag tta 48 Met Gly Val Pro Ile Gly Glu Ile Ile Pro Arg Lys GluIle Glu Leu 1 5 10 15 gaa aac cta tac ggg aaa aaa atc gca atc gac gctctt aat gca atc 96 Glu Asn Leu Tyr Gly Lys Lys Ile Ala Ile Asp Ala LeuAsn Ala Ile 20 25 30 tac caa ttt ttg tcc aca ata aga cag aaa gat gga actcca ctt atg 144 Tyr Gln Phe Leu Ser Thr Ile Arg Gln Lys Asp Gly Thr ProLeu Met 35 40 45 gat tca aag ggt aga ata acc tcc cac cta agc ggg ctc ttttac agg 192 Asp Ser Lys Gly Arg Ile Thr Ser His Leu Ser Gly Leu Phe TyrArg 50 55 60 aca ata aac cta atg gag gct gga ata aaa cct gtg tat gtt tttgat 240 Thr Ile Asn Leu Met Glu Ala Gly Ile Lys Pro Val Tyr Val Phe Asp65 70 75 80 gga gaa cct cca gaa ttc aaa aag aaa gag ctc gaa aaa aga agagaa 288 Gly Glu Pro Pro Glu Phe Lys Lys Lys Glu Leu Glu Lys Arg Arg Glu85 90 95 gcg aga gag gaa gct gaa gaa aag tgg aga gaa gca ctt gaa aaa gga336 Ala Arg Glu Glu Ala Glu Glu Lys Trp Arg Glu Ala Leu Glu Lys Gly 100105 110 gag ata gag gaa gca aga aaa tat gcc caa aga gca acc agg gta aat384 Glu Ile Glu Glu Ala Arg Lys Tyr Ala Gln Arg Ala Thr Arg Val Asn 115120 125 gaa atg ctc atc gag gat gca aaa aaa ctc tta gag ctt atg gga att432 Glu Met Leu Ile Glu Asp Ala Lys Lys Leu Leu Glu Leu Met Gly Ile 130135 140 cct ata gtt caa gca cct agc gag gga gag gcc caa gct gca tat atg480 Pro Ile Val Gln Ala Pro Ser Glu Gly Glu Ala Gln Ala Ala Tyr Met 145150 155 160 gcc gca aag ggg agc gtg tat gca tcg gct agt caa gat tac gattcc 528 Ala Ala Lys Gly Ser Val Tyr Ala Ser Ala Ser Gln Asp Tyr Asp Ser165 170 175 cta ctt ttt gga gct cca aga ctt gtt aga aac tta aca ata acagga 576 Leu Leu Phe Gly Ala Pro Arg Leu Val Arg Asn Leu Thr Ile Thr Gly180 185 190 aaa aga aag ttg cct ggg aaa aat gtc tac gtc gag ata aag cccgag 624 Lys Arg Lys Leu Pro Gly Lys Asn Val Tyr Val Glu Ile Lys Pro Glu195 200 205 ttg ata att ttg gag gaa gta ctc aag gaa tta aag cta aca agagaa 672 Leu Ile Ile Leu Glu Glu Val Leu Lys Glu Leu Lys Leu Thr Arg Glu210 215 220 aag ctc att gaa cta gca atc ctc gtt gga aca gac tac aac ccagga 720 Lys Leu Ile Glu Leu Ala Ile Leu Val Gly Thr Asp Tyr Asn Pro Gly225 230 235 240 gga ata aag ggc ata ggc ctt aaa aaa gct tta gag att gttaga cac 768 Gly Ile Lys Gly Ile Gly Leu Lys Lys Ala Leu Glu Ile Val ArgHis 245 250 255 tca aaa gat ccg cta gca aag ttc caa aag caa agc gat gtggat tta 816 Ser Lys Asp Pro Leu Ala Lys Phe Gln Lys Gln Ser Asp Val AspLeu 260 265 270 tat gca ata aaa gag ttc ttc cta aac cca cca gtc aca gataac tac 864 Tyr Ala Ile Lys Glu Phe Phe Leu Asn Pro Pro Val Thr Asp AsnTyr 275 280 285 aat tta gtg tgg aga gat ccc gac gaa gag gga ata cta aagttc tta 912 Asn Leu Val Trp Arg Asp Pro Asp Glu Glu Gly Ile Leu Lys PheLeu 290 295 300 tgt gac gag cat gac ttt agt gag gaa aga gta aag aat ggatta gag 960 Cys Asp Glu His Asp Phe Ser Glu Glu Arg Val Lys Asn Gly LeuGlu 305 310 315 320 agg ctt aag aag gca atc aaa agt gga aaa caa tca accctt gaa agt 1008 Arg Leu Lys Lys Ala Ile Lys Ser Gly Lys Gln Ser Thr LeuGlu Ser 325 330 335 tgg ttc aag aga taa 1023 Trp Phe Lys Arg 340 79 340PRT Artificial Synthetic 79 Met Gly Val Pro Ile Gly Glu Ile Ile Pro ArgLys Glu Ile Glu Leu 1 5 10 15 Glu Asn Leu Tyr Gly Lys Lys Ile Ala IleAsp Ala Leu Asn Ala Ile 20 25 30 Tyr Gln Phe Leu Ser Thr Ile Arg Gln LysAsp Gly Thr Pro Leu Met 35 40 45 Asp Ser Lys Gly Arg Ile Thr Ser His LeuSer Gly Leu Phe Tyr Arg 50 55 60 Thr Ile Asn Leu Met Glu Ala Gly Ile LysPro Val Tyr Val Phe Asp 65 70 75 80 Gly Glu Pro Pro Glu Phe Lys Lys LysGlu Leu Glu Lys Arg Arg Glu 85 90 95 Ala Arg Glu Glu Ala Glu Glu Lys TrpArg Glu Ala Leu Glu Lys Gly 100 105 110 Glu Ile Glu Glu Ala Arg Lys TyrAla Gln Arg Ala Thr Arg Val Asn 115 120 125 Glu Met Leu Ile Glu Asp AlaLys Lys Leu Leu Glu Leu Met Gly Ile 130 135 140 Pro Ile Val Gln Ala ProSer Glu Gly Glu Ala Gln Ala Ala Tyr Met 145 150 155 160 Ala Ala Lys GlySer Val Tyr Ala Ser Ala Ser Gln Asp Tyr Asp Ser 165 170 175 Leu Leu PheGly Ala Pro Arg Leu Val Arg Asn Leu Thr Ile Thr Gly 180 185 190 Lys ArgLys Leu Pro Gly Lys Asn Val Tyr Val Glu Ile Lys Pro Glu 195 200 205 LeuIle Ile Leu Glu Glu Val Leu Lys Glu Leu Lys Leu Thr Arg Glu 210 215 220Lys Leu Ile Glu Leu Ala Ile Leu Val Gly Thr Asp Tyr Asn Pro Gly 225 230235 240 Gly Ile Lys Gly Ile Gly Leu Lys Lys Ala Leu Glu Ile Val Arg His245 250 255 Ser Lys Asp Pro Leu Ala Lys Phe Gln Lys Gln Ser Asp Val AspLeu 260 265 270 Tyr Ala Ile Lys Glu Phe Phe Leu Asn Pro Pro Val Thr AspAsn Tyr 275 280 285 Asn Leu Val Trp Arg Asp Pro Asp Glu Glu Gly Ile LeuLys Phe Leu 290 295 300 Cys Asp Glu His Asp Phe Ser Glu Glu Arg Val LysAsn Gly Leu Glu 305 310 315 320 Arg Leu Lys Lys Ala Ile Lys Ser Gly LysGln Ser Thr Leu Glu Ser 325 330 335 Trp Phe Lys Arg 340 80 25 DNAArtificial Synthetic 80 gataccatgg gtgtcccaat tggtg 25 81 37 DNAArtificial Synthetic 81 tcgacgtcga cttatctctt gaaccaactt tcaaggg 37 8220 DNA Artificial Synthetic 82 agcgagggag aggcccaagc 20 83 21 DNAArtificial Synthetic 83 gcctatgccc tttattcctc c 21 84 33 DNA ArtificialSynthetic 84 tggtcgctgt ctcgctgaaa gcgagacagc gtg 33 85 30 DNAArtificial Synthetic 85 tgctctctgg tcgctgtctg aaagacagcg 30 86 26 DNAArtificial Synthetic 86 agaaaggaag ggaagaaagc gaaagg 26 87 27 DNAArtificial Synthetic 87 agaaaggaag ggaagaaagc gaaaggc 27 88 24 DNAArtificial Synthetic 88 gccggcgaac gtggcgagaa aggc 24 89 27 DNAArtificial Synthetic 89 agaaaggaag ggaagaaagc gaaaggc 27 90 30 DNAArtificial Synthetic 90 aaaattcctt tctctttgcc ctttgcttcc 30 91 26 DNAArtificial Synthetic 91 ggaaagccgg cgaacgtggc gagaaa 26 92 24 DNAArtificial Synthetic 92 ggaaagccgg cgaacgtggc gaga 24 93 27 DNAArtificial Synthetic 93 agaaaggaag ggaagaaagc gaaaggt 27 94 23 DNAArtificial Synthetic 94 ttccagagcc taatttgcca gta 23 95 23 DNAArtificial Synthetic 95 ttccagagcc taatttgcca gta 23 96 25 DNAArtificial Synthetic 96 cttaccaacg ctaacgagcg tcttg 25 97 43 DNAArtificial Synthetic 97 cccgtctcgc tggtgaaaag aaaaaccacc ctggcgccca ata43 98 23 DNA Artificial Synthetic 98 tattgggcgc catggtggtt ttt 23 99 23DNA Artificial Synthetic 99 tattgggcgn cagggnggtt ttt 23 100 23 DNAArtificial Synthetic 100 tattgggcgn catggnggtt ttt 23 101 23 DNAArtificial Synthetic 101 tattgggcgc cagggnggtt ttt 23 102 23 DNAArtificial Synthetic 102 tattgggcgc catggnggtt ttt 23 103 56 DNAArtificial Synthetic 103 ctgaatataa acttgtggta gttggagctg gtgccgtaggcaagagtgcc ttgacg 56 104 56 DNA Artificial Synthetic 104 ctgaatataaacttgtggta gttggagctg gtgacgtagg caagagtgcc ttgacg 56 105 23 DNAArtificial Synthetic 105 gctcaaggca ctcttgccta cga 23 106 8 DNAArtificial Synthetic 106 ttcaccag 8 107 27 DNA Artificial Synthetic 107ctccaactac cacaagttta tattcag 27 108 14 DNA Artificial Synthetic 108cgagagacca cgct 14 109 14 DNA Artificial Synthetic 109 cgagagacca cgct14 110 14 DNA Artificial Synthetic 110 cgagagacca cgct 14 111 15 DNAArtificial Synthetic 111 cgagagacca cgctg 15 112 30 DNA ArtificialSynthetic 112 gtaatcttac caacgctaac gagcgtcttg 30 113 31 DNA ArtificialSynthetic 113 cctaatttgc cagttacaaa ataaacagcc c 31 114 8 DNA ArtificialSynthetic 114 ttccagag 8 115 44 DNA Artificial Synthetic 115 ttttccagagcctaatgaaa ttaggctctg gaaagacgct cgtg 44 116 14 DNA Artificial Synthetic116 aacgagcgtc tttg 14 117 14 DNA Artificial Synthetic 117 aacgagcgtcattg 14 118 50 DNA Artificial Synthetic 118 ttttttttta attaggctctggaaagacgc tcgtgaaacg agcgtctttg 50 119 17 DNA Artificial Synthetic 119ttttccagag cctaatg 17 120 13 DNA Artificial Synthetic 120 ccggtcgtcc tgg13 121 25 DNA Artificial Synthetic 121 caattccggt gtactcaccg gttcc 25122 16 DNA Artificial Synthetic 122 ccggtcgtcc tggcaa 16 123 47 DNAArtificial Synthetic 123 tgttttgacc tccatagaag accctatagt gagtcgtattaatttcg 47 124 23 DNA Artificial Synthetic 124 cgaaattaat acgactcact ata23 125 30 DNA Artificial Synthetic 125 cgaaattaat acgactcact atacccagaa30 126 16 DNA Artificial Synthetic 126 cgaaattaat acgact 16 127 13 DNAArtificial Synthetic 127 cgaaattaat acg 13 128 12 DNA ArtificialSynthetic 128 cgaaattaat ac 12 129 25 DNA Artificial Synthetic 129cactataggg tcttctatgg aggtc 25 130 28 DNA Artificial Synthetic 130actcactata gggtcttcta tggaggtc 28 131 29 DNA Artificial Synthetic 131gactcactat agggtcttct atggaggtc 29 132 30 DNA Artificial Synthetic 132cgaaattaat acgcagtatg ttagcaaacg 30 133 30 DNA Artificial Synthetic 133gaactggcat gattaagact ccttattacc 30 134 29 DNA Artificial Synthetic 134gaactggcat gattaagact ccttattaa 29 135 326 PRT Artificial Synthetic 135Met Gly Val Gln Phe Gly Asp Phe Ile Pro Lys Asn Ile Ile Ser Phe 1 5 1015 Glu Asp Leu Lys Gly Lys Lys Val Ala Ile Asp Gly Met Asn Ala Leu 20 2530 Tyr Gln Phe Leu Thr Ser Ile Arg Leu Arg Asp Gly Ser Pro Leu Arg 35 4045 Asn Arg Lys Gly Glu Ile Thr Ser Ala Tyr Asn Gly Val Phe Tyr Lys 50 5560 Thr Ile His Leu Leu Glu Asn Asp Ile Thr Pro Ile Trp Val Phe Asp 65 7075 80 Gly Glu Pro Pro Lys Leu Lys Glu Lys Thr Arg Lys Val Arg Arg Glu 8590 95 Met Lys Glu Lys Ala Glu Leu Lys Met Lys Glu Ala Ile Lys Lys Glu100 105 110 Asp Phe Glu Glu Ala Ala Lys Tyr Ala Lys Arg Val Ser Tyr LeuThr 115 120 125 Pro Lys Met Val Glu Asn Cys Lys Tyr Leu Leu Ser Leu MetGly Ile 130 135 140 Pro Tyr Val Glu Ala Pro Ser Glu Gly Glu Ala Gln AlaSer Tyr Met 145 150 155 160 Ala Lys Lys Gly Asp Val Trp Ala Val Val SerGln Asp Tyr Asp Ala 165 170 175 Leu Leu Tyr Gly Ala Pro Arg Val Val ArgAsn Leu Thr Thr Thr Lys 180 185 190 Glu Met Pro Glu Leu Ile Glu Leu AsnGlu Val Leu Glu Asp Leu Arg 195 200 205 Ile Ser Leu Asp Asp Leu Ile AspIle Ala Ile Phe Met Gly Thr Asp 210 215 220 Tyr Asn Pro Gly Gly Val LysGly Ile Gly Phe Lys Arg Ala Tyr Glu 225 230 235 240 Leu Val Arg Ser GlyVal Ala Lys Asp Val Leu Lys Lys Glu Val Glu 245 250 255 Tyr Tyr Asp GluIle Lys Arg Ile Phe Lys Glu Pro Lys Val Thr Asp 260 265 270 Asn Tyr SerLeu Ser Leu Lys Leu Pro Asp Lys Glu Gly Ile Ile Lys 275 280 285 Phe LeuVal Asp Glu Asn Asp Phe Asn Tyr Asp Arg Val Lys Lys His 290 295 300 ValAsp Lys Leu Tyr Asn Leu Ile Ala Asn Lys Thr Lys Gln Lys Thr 305 310 315320 Leu Asp Ala Trp Phe Lys 325 136 340 PRT Artificial Synthetic 136 MetGly Val Pro Ile Gly Glu Ile Ile Pro Arg Lys Glu Ile Glu Leu 1 5 10 15Glu Asn Leu Tyr Gly Lys Lys Ile Ala Ile Asp Ala Leu Asn Ala Ile 20 25 30Tyr Gln Phe Leu Ser Thr Ile Arg Gln Lys Asp Gly Thr Pro Leu Met 35 40 45Asp Ser Lys Gly Arg Ile Thr Ser His Leu Ser Gly Leu Phe Tyr Arg 50 55 60Thr Ile Asn Leu Met Glu Ala Gly Ile Lys Pro Val Tyr Val Phe Asp 65 70 7580 Gly Glu Pro Pro Glu Phe Lys Lys Lys Glu Leu Glu Lys Arg Arg Glu 85 9095 Ala Arg Glu Glu Ala Glu Glu Lys Trp Arg Glu Ala Leu Glu Lys Gly 100105 110 Glu Ile Glu Glu Ala Arg Lys Tyr Ala Gln Arg Ala Thr Arg Val Asn115 120 125 Glu Met Leu Ile Glu Asp Ala Lys Lys Leu Leu Glu Leu Met GlyIle 130 135 140 Pro Ile Val Gln Ala Pro Ser Glu Gly Glu Ala Gln Ala AlaTyr Met 145 150 155 160 Ala Ala Lys Gly Ser Val Tyr Ala Ser Ala Ser GlnAsp Tyr Asp Ser 165 170 175 Leu Leu Phe Gly Ala Pro Arg Leu Val Arg AsnLeu Thr Ile Thr Gly 180 185 190 Lys Arg Lys Leu Pro Gly Lys Asn Val TyrVal Glu Ile Lys Pro Glu 195 200 205 Leu Ile Ile Leu Glu Glu Val Leu LysGlu Leu Lys Leu Thr Arg Glu 210 215 220 Lys Leu Ile Glu Leu Ala Ile LeuVal Gly Thr Asp Tyr Asn Pro Gly 225 230 235 240 Gly Ile Lys Gly Ile GlyLeu Lys Lys Ala Leu Glu Ile Val Arg His 245 250 255 Ser Lys Asp Pro LeuAla Lys Phe Gln Lys Gln Ser Asp Val Asp Leu 260 265 270 Tyr Ala Ile LysGlu Phe Phe Leu Asn Pro Pro Val Thr Asp Asn Tyr 275 280 285 Asn Leu ValTrp Arg Asp Pro Asp Glu Glu Gly Ile Leu Lys Phe Leu 290 295 300 Cys AspGlu His Asp Phe Ser Glu Glu Arg Val Lys Asn Gly Leu Glu 305 310 315 320Arg Leu Lys Lys Ala Ile Lys Ser Gly Lys Gln Ser Thr Leu Glu Ser 325 330335 Trp Phe Lys Arg 340 137 380 PRT Artificial Synthetic 137 Met Gly IleGln Gly Leu Ala Lys Leu Ile Ala Asp Val Ala Pro Ser 1 5 10 15 Ala IleArg Glu Asn Asp Ile Lys Ser Tyr Phe Gly Arg Lys Val Ala 20 25 30 Ile AspAla Ser Met Ser Ile Tyr Gln Phe Leu Ile Ala Val Arg Gln 35 40 45 Gly GlyAsp Val Leu Gln Asn Glu Glu Gly Glu Thr Thr Ser His Leu 50 55 60 Met GlyMet Phe Tyr Arg Thr Ile Arg Met Met Glu Asn Gly Ile Lys 65 70 75 80 ProVal Tyr Val Phe Asp Gly Lys Pro Pro Gln Leu Lys Ser Gly Glu 85 90 95 LeuAla Lys Arg Ser Glu Arg Arg Ala Glu Ala Glu Lys Gln Leu Gln 100 105 110Gln Ala Gln Ala Ala Gly Ala Glu Gln Glu Val Glu Lys Phe Thr Lys 115 120125 Arg Leu Val Lys Val Thr Lys Gln His Asn Asp Glu Cys Lys His Leu 130135 140 Leu Ser Leu Met Gly Ile Pro Tyr Leu Asp Ala Pro Ser Glu Ala Glu145 150 155 160 Ala Ser Cys Ala Ala Leu Val Lys Ala Gly Lys Val Tyr AlaAla Ala 165 170 175 Thr Glu Asp Met Asp Cys Leu Thr Phe Gly Ser Pro ValLeu Met Arg 180 185 190 His Leu Thr Ala Ser Glu Ala Lys Lys Leu Pro IleGln Glu Phe His 195 200 205 Leu Ser Arg Ile Leu Gln Glu Leu Gly Leu AsnGln Glu Gln Phe Val 210 215 220 Asp Leu Cys Ile Leu Leu Gly Ser Asp TyrCys Glu Ser Ile Arg Gly 225 230 235 240 Ile Gly Pro Lys Arg Ala Val AspLeu Ile Gln Lys His Lys Ser Ile 245 250 255 Glu Glu Ile Val Arg Arg LeuAsp Pro Asn Lys Tyr Pro Val Pro Glu 260 265 270 Asn Trp Leu His Lys GluAla His Gln Leu Phe Leu Glu Pro Glu Val 275 280 285 Leu Asp Pro Glu SerVal Glu Leu Lys Trp Ser Glu Pro Asn Glu Glu 290 295 300 Glu Leu Ile LysPhe Met Cys Gly Glu Lys Gln Phe Ser Glu Glu Arg 305 310 315 320 Ile ArgSer Gly Val Lys Arg Leu Ser Lys Ser Arg Gln Gly Ser Thr 325 330 335 GlnGly Arg Leu Asp Asp Phe Phe Lys Val Thr Gly Ser Leu Ser Ser 340 345 350Ala Lys Arg Lys Glu Pro Glu Pro Lys Gly Ser Thr Lys Lys Lys Ala 355 360365 Lys Thr Gly Ala Ala Gly Lys Phe Lys Arg Gly Lys 370 375 380 138 378PRT Artificial Synthetic 138 Met Gly Ile His Gly Leu Ala Lys Leu Ile AlaAsp Val Ala Pro Ser 1 5 10 15 Ala Ile Arg Glu Asn Asp Ile Lys Ser TyrPhe Gly Arg Lys Val Ala 20 25 30 Ile Asp Ala Ser Met Ser Ile Tyr Gln PheLeu Ile Ala Val Arg Gln 35 40 45 Gly Gly Asp Val Leu Gln Asn Glu Glu GlyGlu Thr Thr Ser Leu Met 50 55 60 Gly Met Phe Tyr Arg Thr Ile Arg Met GluAsn Gly Ile Lys Pro Val 65 70 75 80 Tyr Val Phe Asp Gly Lys Pro Pro GlnLeu Lys Ser Gly Glu Leu Ala 85 90 95 Lys Arg Ser Glu Arg Arg Ala Glu AlaGlu Lys Gln Leu Gln Gln Ala 100 105 110 Gln Glu Ala Gly Met Glu Glu GluVal Glu Lys Phe Thr Lys Arg Leu 115 120 125 Val Lys Val Thr Lys Gln HisAsn Asp Glu Cys Lys His Leu Leu Ser 130 135 140 Leu Met Gly Ile Pro TyrLeu Asp Ala Pro Ser Glu Ala Glu Ala Ser 145 150 155 160 Cys Ala Ala LeuAla Lys Ala Gly Lys Val Tyr Ala Ala Ala Thr Glu 165 170 175 Asp Met AspCys Leu Thr Phe Gly Ser Pro Val Leu Met Arg His Leu 180 185 190 Thr AlaSer Glu Ala Lys Lys Leu Pro Ile Gln Glu Phe His Leu Ser 195 200 205 ArgVal Leu Gln Glu Leu Gly Leu Asn Gln Glu Gln Phe Val Asp Leu 210 215 220Cys Ile Leu Leu Gly Ser Asp Tyr Cys Glu Ser Ile Arg Gly Ile Gly 225 230235 240 Ala Lys Arg Ala Val Asp Leu Ile Gln Lys His Lys Ser Ile Glu Glu245 250 255 Ile Val Arg Arg Leu Asp Pro Ser Lys Tyr Pro Val Pro Glu AsnTrp 260 265 270 Leu His Lys Glu Ala Gln Gln Leu Phe Leu Glu Pro Glu ValVal Asp 275 280 285 Pro Glu Ser Val Glu Leu Lys Trp Ser Glu Pro Asn GluGlu Glu Leu 290 295 300 Val Lys Phe Met Cys Gly Glu Lys Gln Phe Ser GluGlu Arg Ile Arg 305 310 315 320 Ser Gly Val Lys Arg Leu Ser Lys Ser ArgGln Gly Ser Thr Gln Gly 325 330 335 Arg Leu Asp Asp Phe Phe Lys Val ThrGly Ser Leu Ser Ser Ala Lys 340 345 350 Arg Lys Glu Pro Glu Pro Lys GlyPro Ala Lys Lys Lys Ala Lys Thr 355 360 365 Gly Gly Ala Gly Lys Phe ArgArg Gly Lys 370 375 139 382 PRT Artificial Synthetic 139 Met Gly Ile LysGly Leu Asn Ala Ile Ile Ser Glu His Val Pro Ser 1 5 10 15 Ala Ile ArgLys Ser Asp Ile Lys Ser Phe Phe Gly Arg Lys Val Ala 20 25 30 Ile Asp AlaSer Met Ser Leu Tyr Gln Phe Leu Ile Ala Val Arg Gln 35 40 45 Gln Asp GlyGly Gln Leu Thr Asn Glu Ala Gly Glu Thr Thr Ser His 50 55 60 Leu Met GlyMet Phe Tyr Arg Thr Leu Arg Met Ile Asp Asn Gly Ile 65 70 75 80 Lys ProCys Tyr Val Phe Asp Gly Lys Pro Pro Asp Leu Lys Ser His 85 90 95 Glu LeuThr Lys Arg Ser Ser Arg Arg Val Glu Thr Glu Lys Lys Leu 100 105 110 AlaGlu Ala Thr Thr Glu Leu Glu Lys Met Lys Gln Glu Arg Arg Leu 115 120 125Val Lys Val Ser Lys Glu His Asn Glu Glu Ala Gln Lys Leu Leu Gly 130 135140 Leu Met Gly Ile Pro Tyr Ile Ile Ala Pro Thr Glu Ala Glu Ala Gln 145150 155 160 Cys Ala Glu Leu Ala Lys Lys Gly Lys Val Tyr Ala Ala Ala SerGlu 165 170 175 Asp Met Asp Thr Leu Cys Tyr Arg Thr Pro Phe Leu Leu ArgHis Leu 180 185 190 Thr Phe Ser Glu Ala Lys Lys Glu Pro Ile His Glu IleAsp Thr Glu 195 200 205 Leu Val Leu Arg Gly Leu Asp Leu Thr Ile Glu GlnPhe Val Asp Leu 210 215 220 Cys Ile Met Leu Gly Cys Asp Tyr Cys Glu SerIle Arg Gly Val Gly 225 230 235 240 Pro Val Thr Ala Leu Lys Leu Ile LysThr His Gly Ser Ile Glu Lys 245 250 255 Ile Val Glu Phe Ile Glu Ser GlyGlu Ser Asn Asn Thr Lys Trp Lys 260 265 270 Ile Pro Glu Asp Trp Pro TyrLys Gln Ala Arg Met Leu Phe Leu Asp 275 280 285 Pro Glu Val Ile Asp GlyAsn Glu Ile Asn Leu Lys Trp Ser Pro Pro 290 295 300 Lys Glu Lys Glu LeuIle Glu Tyr Leu Cys Asp Asp Lys Lys Phe Ser 305 310 315 320 Glu Glu ArgVal Lys Ser Gly Ile Ser Arg Leu Lys Lys Gly Leu Lys 325 330 335 Ser GlyIle Gln Gly Arg Leu Asp Gly Phe Phe Gln Val Val Pro Lys 340 345 350 ThrLys Glu Gln Leu Ala Ala Ala Ala Lys Arg Ala Gln Glu Asn Lys 355 360 365Lys Leu Asn Lys Asn Lys Asn Lys Val Thr Lys Gly Arg Arg 370 375 380 140387 PRT Artificial Synthetic 140 Met Gly Val His Ser Phe Trp Asp Ile AlaGly Pro Thr Ala Arg Pro 1 5 10 15 Val Arg Leu Glu Ser Leu Glu Asp LysArg Met Ala Val Asp Ala Ser 20 25 30 Ile Trp Ile Tyr Gln Phe Leu Lys AlaVal Arg Asp Gln Glu Gly Asn 35 40 45 Ala Val Lys Asn Ser His Ile Thr GlyPhe Phe Arg Arg Ile Cys Lys 50 55 60 Leu Leu Tyr Phe Gly Ile Arg Pro ValPhe Val Phe Asp Gly Gly Val 65 70 75 80 Pro Val Leu Lys Arg Glu Thr IleArg Gln Arg Lys Glu Arg Arg Gln 85 90 95 Gly Lys Arg Glu Ser Ala Lys SerThr Ala Arg Lys Leu Leu Ala Leu 100 105 110 Gln Leu Gln Asn Gly Ser AsnAsp Asn Glu Val Thr Met Asp Met Ile 115 120 125 Lys Glu Val Gln Glu LeuLeu Ser Arg Phe Gly Ile Pro Tyr Ile Thr 130 135 140 Ala Pro Met Glu AlaGlu Ala Gln Cys Ala Glu Leu Leu Gln Leu Asn 145 150 155 160 Leu Val AspGly Ile Ile Thr Asp Asp Ser Asp Val Phe Leu Phe Gly 165 170 175 Gly ThrLys Ile Tyr Lys Asn Met Phe His Glu Lys Asn Tyr Val Glu 180 185 190 PheTyr Asp Ala Glu Ser Ile Leu Lys Leu Leu Gly Leu Asp Arg Lys 195 200 205Asn Met Ile Glu Leu Ala Gln Leu Leu Gly Ser Asp Tyr Thr Asn Gly 210 215220 Leu Lys Gly Met Gly Pro Val Ser Ser Ile Glu Val Ile Ala Glu Phe 225230 235 240 Gly Asn Leu Lys Asn Phe Lys Asp Trp Tyr Asn Asn Gly Gln PheAsp 245 250 255 Lys Arg Lys Gln Glu Thr Glu Asn Lys Phe Glu Lys Asp LeuArg Lys 260 265 270 Lys Leu Val Asn Asn Glu Ile Ile Leu Asp Asp Asp PhePro Ser Val 275 280 285 Met Val Tyr Asp Ala Tyr Met Arg Pro Glu Val AspHis Asp Thr Thr 290 295 300 Pro Phe Val Trp Gly Val Pro Asp Leu Asp MetLeu Arg Ser Phe Met 305 310 315 320 Lys Thr Gln Leu Gly Trp Pro His GluLys Ser Asp Glu Ile Leu Ile 325 330 335 Pro Leu Ile Arg Asp Val Asn LysArg Lys Lys Lys Gly Lys Gln Lys 340 345 350 Arg Ile Asn Glu Phe Phe ProArg Glu Tyr Ile Ser Gly Asp Lys Lys 355 360 365 Leu Asn Thr Ser Lys ArgIle Ser Thr Ala Thr Gly Lys Leu Lys Lys 370 375 380 Arg Lys Met 385 141488 PRT Artificial Synthetic 141 Met Gly Val Ser Gly Leu Trp Asn Ile LeuGlu Pro Val Lys Arg Pro 1 5 10 15 Val Lys Leu Glu Thr Leu Val Asn LysArg Leu Ala Ile Asp Ala Ser 20 25 30 Ile Trp Ile Tyr Gln Phe Leu Lys AlaVal Arg Asp Lys Glu Gly Asn 35 40 45 Gln Leu Lys Ser Ser His Val Val GlyPhe Phe Arg Arg Ile Cys Lys 50 55 60 Leu Leu Phe Phe Gly Ile Lys Pro ValPhe Val Phe Asp Gly Gly Ala 65 70 75 80 Pro Ser Leu Lys Arg Gln Thr IleGln Lys Arg Gln Ala Arg Arg Leu 85 90 95 Asp Arg Glu Glu Asn Ala Thr ValThr Ala Asn Lys Leu Leu Ala Leu 100 105 110 Gln Met Arg His Gln Ala MetLeu Leu Lys Arg Asp Ala Asp Glu Val 115 120 125 Thr Gln Val Met Ile LysGlu Cys Gln Glu Leu Leu Arg Leu Phe Gly 130 135 140 Leu Pro Tyr Ile ValAla Pro Gln Glu Ala Glu Ala Gln Cys Ser Lys 145 150 155 160 Leu Leu GluLeu Lys Leu Val Asp Gly Ile Val Thr Asp Asp Ser Asp 165 170 175 Val PheLeu Phe Gly Gly Thr Arg Val Tyr Arg Asn Met Phe Asn Gln 180 185 190 AsnLys Phe Val Glu Leu Tyr Leu Met Asp Asp Met Lys Arg Glu Phe 195 200 205Asn Val Asn Gln Met Asp Leu Ile Lys Leu Ala His Leu Leu Gly Ser 210 215220 Asp Tyr Thr Met Gly Leu Ser Arg Val Gly Pro Val Leu Ala Leu Glu 225230 235 240 Ile Leu His Glu Phe Pro Gly Asp Thr Gly Leu Phe Glu Phe LysLys 245 250 255 Trp Phe Gln Arg Leu Ser Thr Gly His Ala Ser Lys Asn AspVal Asn 260 265 270 Thr Pro Val Lys Lys Arg Ile Asn Lys Leu Val Gly LysIle Ile Leu 275 280 285 Pro Ser Glu Phe Pro Asn Pro Leu Val Asp Glu AlaTyr Leu His Pro 290 295 300 Ala Val Asp Asp Ser Lys Gln Ser Phe Gln TrpGly Ile Pro Asp Leu 305 310 315 320 Asp Glu Leu Arg Gln Phe Leu Met AlaThr Val Gly Trp Ser Lys Gln 325 330 335 Arg Thr Asn Glu Val Leu Leu ProVal Ile Gln Asp Met His Lys Lys 340 345 350 Gln Phe Val Gly Thr Gln SerAsn Leu Thr Gln Phe Phe Glu Gly Gly 355 360 365 Asn Thr Asn Val Tyr AlaPro Arg Val Ala Tyr His Phe Lys Ser Lys 370 375 380 Arg Leu Glu Asn AlaLeu Ser Ser Phe Lys Asn Gln Ile Ser Asn Gln 385 390 395 400 Ser Pro MetSer Glu Glu Ile Gln Ala Asp Ala Asp Ala Phe Gly Glu 405 410 415 Ser LysGly Ser Asp Glu Leu Gln Ser Arg Ile Leu Arg Arg Lys Lys 420 425 430 MetMet Ala Ser Lys Asn Ser Ser Asp Ser Asp Ser Asp Ser Glu Asp 435 440 445Asn Phe Leu Ala Ser Leu Thr Pro Lys Thr Asn Ser Ser Ser Ile Ser 450 455460 Ile Glu Asn Leu Pro Arg Lys Thr Lys Leu Ser Thr Ser Leu Leu Lys 465470 475 480 Lys Pro Ser Lys Arg Arg Arg Lys 485 142 550 PRT ArtificialSynthetic 142 Met Gly Val Gln Gly Leu Trp Lys Leu Leu Glu Cys Ser GlyArg Gln 1 5 10 15 Val Ser Pro Glu Ala Leu Glu Gly Lys Ile Leu Ala ValAsp Ile Ser 20 25 30 Ile Trp Leu Asn Gln Ala Leu Lys Gly Val Arg Asp ArgHis Gly Asn 35 40 45 Ser Ile Glu Asn Pro His Leu Leu Thr Leu Phe His ArgLeu Cys Lys 50 55 60 Leu Leu Phe Phe Arg Ile Arg Pro Ile Phe Val Phe AspGly Asp Ala 65 70 75 80 Pro Leu Leu Lys Lys Gln Thr Leu Val Lys Arg ArgGln Arg Lys Asp 85 90 95 Leu Ala Ser Ser Asp Ser Arg Lys Thr Thr Glu LysLeu Leu Lys Thr 100 105 110 Phe Leu Lys Arg Gln Ala Ile Lys Thr Glu ArgIle Ala Ala Thr Val 115 120 125 Thr Gly Gln Met Phe Leu Glu Ser Gln GluLeu Leu Arg Leu Phe Gly 130 135 140 Ile Pro Tyr Ile Gln Ala Pro Met GluAla Glu Ala Gln Cys Ala Ile 145 150 155 160 Leu Asp Leu Thr Asp Gln ThrSer Gly Thr Ile Thr Asp Asp Ser Asp 165 170 175 Ile Trp Leu Phe Gly AlaArg His Val Tyr Arg Asn Phe Phe Asn Lys 180 185 190 Asn Lys Phe Val GluTyr Tyr Gln Tyr Val Asp Phe His Asn Gln Leu 195 200 205 Gly Leu Asp ArgAsn Lys Leu Ile Asn Leu Ala Tyr Leu Leu Gly Ser 210 215 220 Asp Tyr ThrGlu Gly Ile Pro Thr Val Gly Cys Val Thr Ala Met Glu 225 230 235 240 IleLeu Asn Glu Phe Pro Gly His Gly Leu Glu Pro Leu Leu Lys Phe 245 250 255Ser Glu Trp Trp His Glu Ala Gln Lys Asn Pro Lys Ile Arg Pro Asn 260 265270 Pro His Asp Thr Lys Val Lys Lys Lys Leu Arg Thr Leu Gln Leu Thr 275280 285 Pro Gly Phe Pro Asn Pro Ala Val Ala Glu Ala Tyr Leu Lys Pro Val290 295 300 Val Asp Asp Ser Lys Gly Ser Phe Leu Trp Gly Lys Pro Asp LeuAsp 305 310 315 320 Lys Ile Arg Glu Phe Cys Gln Arg Tyr Phe Gly Trp AsnArg Thr Lys 325 330 335 Thr Asp Glu Ser Leu Phe Pro Val Leu Lys Gln LeuAsp Ala Gln Gln 340 345 350 Thr Gln Leu Arg Ile Asp Ser Phe Phe Arg LeuAla Gln Gln Glu Lys 355 360 365 Glu Asp Ala Lys Arg Ile Lys Ser Gln ArgLeu Asn Arg Ala Val Thr 370 375 380 Cys Met Leu Arg Lys Glu Lys Glu AlaAla Ala Ser Glu Ile Glu Ala 385 390 395 400 Val Ser Val Ala Met Glu LysGlu Phe Glu Leu Leu Asp Lys Ala Lys 405 410 415 Arg Lys Thr Gln Lys ArgGly Ile Thr Asn Thr Leu Glu Glu Ser Ser 420 425 430 Ser Leu Lys Arg LysArg Leu Ser Asp Ser Lys Arg Lys Asn Thr Cys 435 440 445 Gly Gly Phe LeuGly Glu Thr Cys Leu Ser Glu Ser Ser Asp Gly Ser 450 455 460 Ser Ser GluHis Ala Glu Ser Ser Ser Leu Met Asn Val Gln Arg Arg 465 470 475 480 ThrAla Ala Lys Glu Pro Lys Thr Ser Ala Ser Asp Ser Gln Asn Ser 485 490 495Val Lys Glu Ala Pro Val Lys Asn Gly Gly Ala Thr Thr Ser Ser Ser 500 505510 Ser Asp Ser Asp Asp Asp Gly Gly Lys Glu Lys Met Val Leu Val Thr 515520 525 Ala Arg Ser Val Phe Gly Lys Lys Arg Arg Lys Leu Arg Arg Ala Arg530 535 540 Gly Arg Lys Arg Lys Thr 545 550 143 543 PRT ArtificialSynthetic 143 Met Gly Val Gln Gly Leu Trp Lys Leu Leu Glu Cys Ser GlyHis Arg 1 5 10 15 Val Ser Pro Glu Ala Leu Glu Gly Lys Val Leu Ala ValAsp Ile Ser 20 25 30 Ile Trp Leu Asn Gln Ala Leu Lys Gly Val Arg Asp SerHis Gly Asn 35 40 45 Val Ile Glu Asn Ala His Leu Leu Thr Leu Phe His ArgLeu Cys Lys 50 55 60 Leu Leu Phe Phe Arg Ile Arg Pro Ile Phe Val Phe AspGly Asp Ala 65 70 75 80 Pro Leu Leu Lys Lys Gln Thr Leu Ala Lys Arg ArgGln Arg Lys Asp 85 90 95 Ser Ala Ser Ile Asp Ser Arg Lys Thr Thr Glu LysLeu Leu Lys Thr 100 105 110 Phe Leu Lys Arg Gln Ala Leu Lys Thr Asp ArgIle Ala Ala Ser Val 115 120 125 Thr Gly Gln Met Phe Leu Glu Ser Gln GluLeu Leu Arg Leu Phe Gly 130 135 140 Val Pro Tyr Ile Gln Ala Pro Met GluAla Glu Ala Gln Cys Ala Val 145 150 155 160 Leu Asp Leu Ser Asp Gln ThrSer Gly Thr Ile Thr Asp Asp Ser Asp 165 170 175 Ile Trp Leu Phe Gly AlaArg His Val Tyr Lys Asn Phe Phe Asn Lys 180 185 190 Asn Lys Phe Val GluTyr Tyr Gln Tyr Val Asp Phe Tyr Ser Gln Leu 195 200 205 Gly Leu Asp ArgAsn Lys Leu Ile Asn Leu Ala Tyr Leu Leu Gly Ser 210 215 220 Asp Tyr ThrGlu Gly Ile Pro Thr Val Gly Cys Val Thr Ala Met Glu 225 230 235 240 IleLeu Asn Glu Phe Pro Gly Arg Gly Leu Asp Pro Leu Leu Lys Phe 245 250 255Ser Glu Trp Trp His Glu Ala Gln Asn Asn Lys Lys Val Ala Glu Asn 260 265270 Pro Tyr Asp Thr Lys Val Lys Lys Lys Leu Arg Lys Leu Gln Leu Thr 275280 285 Pro Gly Phe Pro Asn Pro Ala Val Ala Asp Ala Tyr Leu Arg Pro Val290 295 300 Val Asp Asp Ser Arg Gly Ser Phe Leu Trp Gly Lys Pro Asp ValAsp 305 310 315 320 Lys Ile Arg Glu Phe Cys Gln Arg Tyr Phe Gly Trp AsnArg Met Lys 325 330 335 Thr Asp Glu Ser Leu Tyr Pro Val Leu Lys His LeuAsn Ala His Gln 340 345 350 Thr Gln Leu Arg Ile Asp Ser Phe Phe Arg LeuAla Gln Gln Glu Lys 355 360 365 Gln Asp Ala Lys Leu Ile Lys Ser His ArgLeu Ser Arg Ala Val Thr 370 375 380 Cys Met Leu Arg Lys Glu Arg Glu GluLys Ala Pro Glu Leu Thr Lys 385 390 395 400 Val Thr Glu Ala Met Glu LysGlu Phe Glu Leu Leu Asp Asp Ala Lys 405 410 415 Gly Lys Thr Gln Lys ArgGlu Leu Pro Tyr Lys Lys Glu Thr Ser Val 420 425 430 Pro Lys Arg Arg ArgPro Ser Gly Asn Gly Gly Phe Leu Gly Asp Pro 435 440 445 Tyr Cys Ser GluSer Pro Gln Glu Ser Ser Cys Glu Asp Gly Glu Gly 450 455 460 Ser Ser ValMet Ser Ala Arg Gln Arg Ser Ala Ala Glu Ser Ser Lys 465 470 475 480 IleGly Cys Ser Asp Val Pro Asp Leu Val Arg Asp Ser Pro His Gly 485 490 495Arg Gln Gly Cys Val Ser Thr Ser Ser Ser Asp Ser Glu Asp Gly Glu 500 505510 Asp Lys Ala Lys Thr Val Leu Val Thr Ala Arg Pro Val Phe Gly Lys 515520 525 Lys Arg Arg Lys Leu Lys Ser Met Lys Arg Arg Lys Lys Lys Thr 530535 540 144 527 PRT Artificial Synthetic 144 Met Gly Val Gln Gly Leu TrpLys Leu Leu Glu Cys Ser Gly Arg Pro 1 5 10 15 Ile Asn Pro Gly Thr LeuGlu Gly Lys Ile Leu Ala Val Asp Ile Ser 20 25 30 Ile Trp Leu Asn Gln AlaVal Lys Gly Ala Arg Asp Arg Gln Gly Asn 35 40 45 Ala Ile Gln Asn Ala HisLeu Leu Thr Leu Phe His Arg Leu Cys Lys 50 55 60 Leu Leu Phe Phe Arg IleArg Pro Ile Phe Val Phe Asp Gly Glu Ala 65 70 75 80 Pro Leu Leu Lys ArgGln Thr Leu Ala Lys Arg Arg Gln Arg Thr Asp 85 90 95 Lys Ala Ser Asn AspAla Arg Lys Thr Asn Glu Lys Leu Leu Arg Thr 100 105 110 Phe Leu Lys ArgGln Ala Ile Lys Ala Glu Arg Ile Ala Ala Thr Val 115 120 125 Thr Gly GlnMet Cys Leu Glu Ser Gln Glu Leu Leu Gln Leu Phe Gly 130 135 140 Ile ProTyr Ile Val Ala Pro Met Glu Ala Glu Ala Gln Cys Ala Ile 145 150 155 160Leu Asp Leu Thr Asp Gln Thr Ser Gly Thr Ile Thr Asp Asp Ser Asp 165 170175 Ile Trp Leu Phe Gly Ala Arg His Val Tyr Lys Asn Phe Phe Ser Gln 180185 190 Asn Lys His Val Glu Tyr Tyr Gln Tyr Ala Asp Ile His Asn Gln Leu195 200 205 Gly Leu Asp Arg Ser Lys Leu Ile Asn Leu Ala Tyr Leu Leu GlySer 210 215 220 Asp Tyr Thr Glu Gly Ile Pro Thr Val Gly Tyr Val Ser AlaMet Glu 225 230 235 240 Ile Leu Asn Glu Phe Pro Gly Gln Gly Leu Glu ProLeu Val Lys Phe 245 250 255 Lys Glu Trp Trp Ser Glu Ala Gln Lys Asp LysLys Met Arg Pro Asn 260 265 270 Pro Asn Asp Thr Lys Val Lys Lys Lys LeuArg Leu Leu Asp Leu Gln 275 280 285 Gln Ser Phe Pro Asn Pro Ala Val AlaSer Ala Tyr Leu Lys Pro Val 290 295 300 Val Asp Glu Ser Lys Ser Ala PheSer Trp Gly Arg Pro Asp Leu Glu 305 310 315 320 Gln Ile Arg Glu Phe CysGlu Ser Arg Phe Gly Trp Tyr Arg Leu Lys 325 330 335 Thr Asp Glu Val LeuLeu Pro Val Leu Lys Gln Leu Asn Ala Gln Gln 340 345 350 Thr Gln Leu ArgIle Asp Ser Phe Phe Arg Leu Glu Gln His Glu Ala 355 360 365 Ala Gly LeuLys Ser Gln Arg Leu Arg Arg Ala Val Thr Cys Met Lys 370 375 380 Arg LysGlu Arg Asp Val Glu Ala Glu Glu Val Glu Ala Ala Val Ala 385 390 395 400Val Met Glu Arg Glu Cys Thr Asn Gln Arg Lys Gly Gln Lys Thr Asn 405 410415 Thr Lys Ser Gln Gly Thr Lys Arg Arg Lys Pro Thr Glu Cys Ser Gln 420425 430 Glu Asp Gln Asp Pro Gly Gly Gly Phe Ile Gly Ile Glu Leu Lys Thr435 440 445 Leu Ser Ser Lys Ala Tyr Ser Ser Asp Gly Ser Ser Ser Asp AlaGlu 450 455 460 Asp Leu Pro Ser Gly Leu Ile Asp Lys Gln Ser Gln Ser GlyIle Val 465 470 475 480 Gly Arg Gln Lys Ala Ser Asn Lys Val Glu Ser SerSer Ser Ser Asp 485 490 495 Asp Glu Asp Arg Thr Val Met Val Thr Ala LysPro Val Phe Gln Gly 500 505 510 Lys Lys Thr Lys Ser Lys Thr Met Lys GluThr Val Lys Arg Lys 515 520 525 145 434 PRT Artificial Synthetic 145 MetThr Ile Asn Gly Ile Trp Glu Trp Ala Asn His Val Val Arg Lys 1 5 10 15Val Pro Asn Glu Thr Met Arg Asp Lys Thr Leu Ser Ile Asp Gly His 20 25 30Ile Trp Leu Tyr Glu Ser Leu Lys Gly Cys Glu Ala His His Gln Gln 35 40 45Thr Pro Asn Ser Tyr Leu Val Thr Phe Phe Thr Arg Ile Gln Arg Leu 50 55 60Leu Glu Leu Lys Ile Ile Pro Ile Val Val Phe Asp Asn Ile Asn Ala 65 70 7580 Ser Ser Ser Ala His Glu Ser Lys Asp Gln Asn Glu Phe Val Pro Arg 85 9095 Lys Arg Arg Ser Phe Gly Asp Ser Pro Phe Thr Asn Leu Val Asp His 100105 110 Val Tyr Lys Thr Asn Ala Leu Leu Thr Glu Leu Gly Ile Lys Val Ile115 120 125 Ile Ala Pro Gly Asp Gly Glu Ala Gln Cys Ala Arg Leu Glu AspLeu 130 135 140 Gly Val Thr Ser Gly Cys Ile Thr Thr Asp Phe Asp Tyr PheLeu Phe 145 150 155 160 Gly Gly Lys Asn Leu Tyr Arg Phe Asp Phe Thr AlaGly Thr Ser Ser 165 170 175 Thr Ala Cys Leu His Asp Ile Met His Leu SerLeu Gly Arg Met Phe 180 185 190 Met Glu Lys Lys Val Ser Arg Pro His LeuIle Ser Thr Ala Ile Leu 195 200 205 Leu Gly Cys Asp Tyr Phe Gln Arg GlyVal Gln Asn Ile Gly Ile Val 210 215 220 Ser Val Phe Asp Ile Leu Gly GluPhe Gly Asp Asp Gly Asn Glu Glu 225 230 235 240 Ile Asp Pro His Val IleLeu Asp Arg Phe Ala Ser Tyr Val Arg Glu 245 250 255 Glu Ile Pro Ala ArgSer Glu Asp Thr Gln Arg Lys Leu Arg Leu Arg 260 265 270 Arg Lys Lys TyrAsn Phe Pro Val Gly Phe Pro Asn Cys Asp Ala Val 275 280 285 His Asn AlaIle Thr Met Tyr Leu Arg Pro Pro Val Ser Ser Glu Ile 290 295 300 Pro LysIle Ile Pro Arg Ala Ala Asn Phe Gln Gln Val Ala Glu Ile 305 310 315 320Met Met Lys Glu Cys Gly Trp Pro Ala Thr Arg Thr Gln Lys Glu Leu 325 330335 Ala Leu Ser Ile Arg Arg Lys Val His Leu Thr Thr Thr Val Ala Gln 340345 350 Thr Arg Ile Pro Asp Phe Phe Ala Ala Thr Lys Ser Lys Asn Phe Thr355 360 365 Pro Ile Val Glu Pro Cys Glu Ser Leu Glu Asp Tyr Ile Ser AlaAsn 370 375 380 Asn Thr Trp Met Arg Lys Arg Lys Arg Ser Glu Ser Pro GlnIle Leu 385 390 395 400 Gln His His Ala Lys Arg Gln Val Pro Asp Arg LysArg Ser Val Lys 405 410 415 Ile Arg Ala Phe Lys Pro Tyr Pro Thr Asp ValIle Glu Leu Gly Asp 420 425 430 Ser Asp 146 33 DNA Artificial Synthetic146 tactgactca ctatagggtc ttctatggag gtc 33 147 42 DNA ArtificialSynthetic 147 ttttttttta attaggctct ggaagacgct gaaagcgtct tg 42 148 38DNA Artificial Synthetic 148 ttttttttta attaggctct ggaagacgga acgtcttg38 149 34 DNA Artificial Synthetic 149 ttttttttta attaggctct ggaagagaatcttg 34 150 32 DNA Artificial Synthetic 150 ttttttttta attaggctctggaaggaact tg 32 151 25 DNA Artificial Synthetic 151 tttttttttaattaggctct ggaag 25 152 26 DNA Artificial Synthetic 152 attagaaaggaagggaagaa agcgaa 26 153 30 DNA Artificial Synthetic 153 acggggaaagccggcgaacg tggcgagaaa 30 154 30 DNA Artificial Synthetic 154 tgacggggaaagccggcgaa cgtggcgaga 30 155 30 DNA Artificial Synthetic 155 cttgacggggaaagccggcg aacgtggcga 30 156 30 DNA Artificial Synthetic 156 gcttgacggggaaagccggc gaacgtggcg 30 157 18 DNA Artificial Synthetic 157 agaaaggaagggaagaaa 18 158 45 DNA Artificial Synthetic 158 tggaggtcaa aacatcgataagtcgaagaa aggaagggaa gaaat 45 159 14 DNA Artificial Synthetic 159tgttttgacc tcca 14 160 30 DNA Artificial Synthetic 160 acacagtgtcctcccgctcc tcctgagcaa 30 161 18 DNA Artificial Synthetic 161 tttccctcctcctcttcc 18 162 54 DNA Artificial Synthetic 162 atgaggaaga ggaggagggtgctcaggagg agcgggagga cactgtgtct gtca 54 163 53 DNA Artificial Synthetic163 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agc 53 1641011 DNA Artificial Synthetic 164 atgggtgcgg atattggtga cctctttgagagggaagagg tcgagcttga gtacttctca 60 ggaaagaaaa ttgccgttga tgctttcaacacgctatacc agttcatctc gataataagg 120 cagcctgacg gtacgccgtt aaaggactcacagggcagaa tcacctctca cctttccgga 180 atcctataca gagtctccaa catggtcgaggtgggaatca ggccggtgtt tgtattcgac 240 ggagagccac cggagttcaa gaaggctgaaattgaggaga ggaaaaagag aagggctgag 300 gcagaggaga tgtggattgc ggctttgcaggcaggagata aggacgcgaa aaagtatgct 360 caggctgcag ggagggttga cgagtacattgttgactccg caaagacgct tttaagttac 420 atggggattc cctttgtcga tgccccgtctgaaggagagg cgcaggctgc ttacatggca 480 gcaaaaggcg atgtggagta cacaggaagccaggattacg attctctgct cttcggaagc 540 ccgagactcg ccagaaatct cgcaataacgggaaaaagga agcttcccgg caaaaatgtc 600 tatgtggatg taaagccgga gataataattctggaaagca acctcaaaag gctgggtttg 660 acgagggagc agctcatcga catagcgattctggtcggga cggactacaa tgagggtgtg 720 aagggtgtcg gcgtcaagaa ggctttgaactacatcaaga cctacggaga tattttcagg 780 gcactcaagg ctctgaaagt aaatattgaccacgtagagg agataaggaa tttcttcctg 840 aatcctcctg tgactgacga ctacagaatagagttcaggg agcctgactt tgagaaggcc 900 atcgagttcc tgtgcgagga gcacgacttcagcagggaga gggtcgagaa ggccttggag 960 aagctcaaag ctctgaagtc aacccaggccacgcttgaga ggtggttctg a 1011 165 336 PRT Artificial Synthetic 165 MetGly Ala Asp Ile Gly Asp Leu Phe Glu Arg Glu Glu Val Glu Leu 1 5 10 15Glu Tyr Phe Ser Gly Lys Lys Ile Ala Val Asp Ala Phe Asn Thr Leu 20 25 30Tyr Gln Phe Ile Ser Ile Ile Arg Gln Pro Asp Gly Thr Pro Leu Lys 35 40 45Asp Ser Gln Gly Arg Ile Thr Ser His Leu Ser Gly Ile Leu Tyr Arg 50 55 60Val Ser Asn Met Val Glu Val Gly Ile Arg Pro Val Phe Val Phe Asp 65 70 7580 Gly Glu Pro Pro Glu Phe Lys Lys Ala Glu Ile Glu Glu Arg Lys Lys 85 9095 Arg Arg Ala Glu Ala Glu Glu Met Trp Ile Ala Ala Leu Gln Ala Gly 100105 110 Asp Lys Asp Ala Lys Lys Tyr Ala Gln Ala Ala Gly Arg Val Asp Glu115 120 125 Tyr Ile Val Asp Ser Ala Lys Thr Leu Leu Ser Tyr Met Gly IlePro 130 135 140 Phe Val Asp Ala Pro Ser Glu Gly Glu Ala Gln Ala Ala TyrMet Ala 145 150 155 160 Ala Lys Gly Asp Val Glu Tyr Thr Gly Ser Gln AspTyr Asp Ser Leu 165 170 175 Leu Phe Gly Ser Pro Arg Leu Ala Arg Asn LeuAla Ile Thr Gly Lys 180 185 190 Arg Lys Leu Pro Gly Lys Asn Val Tyr ValAsp Val Lys Pro Glu Ile 195 200 205 Ile Ile Leu Glu Ser Asn Leu Lys ArgLeu Gly Leu Thr Arg Glu Gln 210 215 220 Leu Ile Asp Ile Ala Ile Leu ValGly Thr Asp Tyr Asn Glu Gly Val 225 230 235 240 Lys Gly Val Gly Val LysLys Ala Leu Asn Tyr Ile Lys Thr Tyr Gly 245 250 255 Asp Ile Phe Arg AlaLeu Lys Ala Leu Lys Val Asn Ile Asp His Val 260 265 270 Glu Glu Ile ArgAsn Phe Phe Leu Asn Pro Pro Val Thr Asp Asp Tyr 275 280 285 Arg Ile GluPhe Arg Glu Pro Asp Phe Glu Lys Ala Ile Glu Phe Leu 290 295 300 Cys GluGlu His Asp Phe Ser Arg Glu Arg Val Glu Lys Ala Leu Glu 305 310 315 320Lys Leu Lys Ala Leu Lys Ser Thr Gln Ala Thr Leu Glu Arg Trp Phe 325 330335 166 26 DNA Artificial Synthetic 166 ccgtcaacat ttaccatggg tgcgga 26167 31 DNA Artificial Synthetic 167 ccgccacctc gtagtcgaca tccttttcgt g31 168 20 DNA Artificial Synthetic 168 ggcgaccaca cccgtcctgt 20 169 20DNA Artificial Synthetic 169 ccacgatgcg tccggcgtag 20 170 29 DNAArtificial Synthetic 170 aacgaggcgc acccacccaa ggcacagcn 29 171 26 DNAArtificial Synthetic 171 acgggtcaat gtccatgccc caaaga 26 172 28 DNAArtificial Synthetic 172 gtctgagatg aaagtgcgcc tcgttaan 28 173 26 DNAArtificial Synthetic 173 tcttcgcaca tttcatctca gacgga 26 174 22 DNAArtificial Synthetic 174 gctgtgcctt gggtgggtgc gn 22 175 29 DNAArtificial Synthetic 175 aacgaggcgc acccacccaa ggcacagcn 29 176 26 DNAArtificial Synthetic 176 acgggtcaat gtccatgccc caaaga 26 177 28 DNAArtificial Synthetic 177 gtctgagatg aaagtgcgcc tcgttaan 28 178 23 DNAArtificial Synthetic 178 tcttcgcaca tttcatctca gac 23 179 18 DNAArtificial Synthetic 179 gctgtgcctt gggtgggn 18 180 20 DNA ArtificialSynthetic 180 gctgtgcctt gggtgggtgn 20 181 22 DNA Artificial Synthetic181 gctgtgcctt gggtgggtgc gn 22 182 23 DNA Artificial Synthetic 182gctgtgcctt gggtgggtgc gcn 23 183 42 DNA Artificial Synthetic 183gtctgagatg aaagtgctcc cgcacccacc caaggcacag cn 42 184 18 DNA ArtificialSynthetic 184 gctgtgcctt gggtgggn 18 185 29 DNA Artificial Synthetic 185aacgaggcgc acccacccaa ggcacagcn 29 186 21 DNA Artificial Synthetic 186gctgtgcctt gggtgggtgc g 21 187 21 DNA Artificial Synthetic 187gctgtgcctt gggtgggtgc n 21 188 21 DNA Artificial Synthetic 188gctgtgcctt gggtgggtgc n 21 189 21 DNA Artificial Synthetic 189gctgtgcctt gggtgggtgc n 21 190 26 DNA Artificial Synthetic 190tcttcgcaca tttcatctca gacgga 26 191 54 DNA Artificial Synthetic 191ctgggcgcgg acatggagga cgtgcgcggc cgcctggtgc agtaccgcgg cgag 54 192 54DNA Artificial Synthetic 192 ctgggcgcgg acatggagga cgtgtgcggc cgcctggtgcagtaccgcgg cgag 54 193 56 DNA Artificial Synthetic 193 cgcgatgccgatgacctgca gaagcgcctg gcagtgtacc aggccggggc ccgcga 56 194 56 DNAArtificial Synthetic 194 cgcgatgccg atgacctgca gaagtgcctg gcagtgtaccaggccggggc ccgcga 56 195 23 DNA Artificial cggtactgcaccaggcggccgct 195cggtactgca ccaggcggcc gct 23 196 25 DNA Artificial Synthetic 196ccccggcctg gtacactgcc aggct 25 197 27 DNA Artificial Synthetic 197aacgaggcgc acgcacgtcc tccatgt 27 198 27 DNA Artificial Synthetic 198gaacgaggcg cacacacgtc ctcctgt 27 199 28 DNA Artificial Synthetic 199aacgaggcgc acgcttctgc aggtcatc 28 200 27 DNA Artificial Synthetic 200aacgaggcgc acacttctgc ggtcatc 27 201 39 DNA Artificial Synthetic 201cctcgtctcg gttttccgag acgagggtgc gcctcgttc 39 202 28 DNA ArtificialSynthetic 202 cccctgggga agagcagaga tatacgtc 28 203 24 DNA ArtificialSynthetic 203 gggctccaca cggcgactct catt 24 204 39 DNA ArtificialSynthetic 204 ggtgctccac ctggcacgta tatctctgct cttccccag 39 205 39 DNAArtificial Synthetic 205 ggtgctccac ctggtacgta tatctctgct cttccccag 39206 46 DNA Artificial Synthetic 206 agctgttcgt gttctatgat catgagagtcgccgtgtgga gccccg 46 207 46 DNA Artificial Synthetic 207 agctgttcgtgttctatgat gatgagagtc gccgtgtgga gccccg 46 208 29 DNA ArtificialSynthetic 208 aacgaacgcg caggccaggt ggagcattt 29 209 27 DNA ArtificialSynthetic 209 aacgaacgcg cagaccaggt ggagcac 27 210 40 DNA ArtificialSynthetic 210 ctccgtctcg gttttccgag acggagctgc gcgttcguuu 40 211 35 DNAArtificial Synthetic 211 aagcacgcag cacgatcata gaacacgaac agttt 35 21235 DNA Artificial Synthetic 212 aagcacgcag caccatcata gaacacgaac agttt35 213 40 DNA Artificial Synthetic 213 acgcgtctcg gttttccgag acgcgtgtgctgcgtgcuuu 40 214 50 DNA Artificial Synthetic 214 gaaggtgtct gcgggagccgatttcatcat cacgcagctt ttctttgagg 50 215 50 DNA Artificial Synthetic 215gaaggtgtct gcgggagtcg atttcatcat cacgcagctt ttctttgagg 50 216 30 DNAArtificial Synthetic 216 caaagaaaag ctgcgtgatg atgaaatcgc 30 217 26 DNAArtificial Synthetic 217 aacgaggcgc acgctcccgc agacac 26 218 27 DNAArtificial Synthetic 218 aacgaggcgc acactcccgc agacacc 27 219 39 DNAArtificial Synthetic 219 cctcgtctcg gttttccgag acgagggtgc gcctcgttt 39220 51 DNA Artificial Synthetic 220 actgggagca ttgaggctcg ctgagagtcacttttattgg gaaccatagt t 51 221 51 DNA Artificial Synthetic 221actgggagca ttgaggcttg ctgagagtca cttttattgg gaaccatagt t 51 222 30 DNAArtificial Synthetic 222 tatggttccc aataaaagtg actctcagct 30 223 28 DNAArtificial Synthetic 223 aacgaggcgc acgagcctca atgctccc 28 224 28 DNAArtificial Synthetic 224 aacgaggcgc acaagcctca atgctccc 28 225 39 DNAArtificial Synthetic 225 cctcgtctcg gttttccgag acgagggtgc gcctcgttt 39226 52 DNA Artificial Synthetic 226 tgaagtctag agaaagggtt gtacggctgaggtctggaga aatgggcatc tg 52 227 46 DNA Artificial Synthetic 227tttgaaatgt cacagggttc ctaacagcca ctcttccctg gatggg 46 228 28 DNAArtificial Synthetic 228 agatgcccat ttctccagac ctcagccc 28 229 36 DNAArtificial Synthetic 229 aagcacgcag cacgtacaac cctttctcta gacaaa 36 23040 DNA Artificial Synthetic 230 ctccgtctcg gttttccgag acggaggtgctgcgtgcuuu 40 231 25 DNA Artificial Synthetic 231 ccatccaggg aagagtggcctgttt 25 232 29 DNA Artificial Synthetic 232 aagcacgcag cacaggaaccctgtgacat 29 233 46 DNA Artificial Synthetic 233 taggttttga ggggcatggggacggggttc agcctccagg gtccta 46 234 46 DNA Artificial Synthetic 234taggttttga ggggcatgag gacggggttc agcctccagg gtccta 46 235 25 DNAArtificial Synthetic 235 gaccctggag gctgaacccc gtcca 25 236 28 DNAArtificial Synthetic 236 aacgaggcgc acccatcggg gtcaaaac 28 237 28 DNAArtificial Synthetic 237 aacgaggcgc actcatgccc ctcaaaac 28 238 56 DNAArtificial Synthetic 238 aaggacaaaa tacctgtatt cctcgcctgt ccagggatctgctcttacag attaga 56 239 56 DNA Artificial Synthetic 239 aaggacaaaatacctgtatt ccttgcctgt ccagggatct gctcttacag attaga 56 240 30 DNAArtificial Synthetic 240 taatctgtaa gagcagatcc ctggacagcc 30 241 33 DNAArtificial Synthetic 241 aacgaggcgc acgaggaata caggtatttt gtc 33 242 33DNA Artificial Synthetic 242 aacgaggcgc acaaggaata caggtatttt gtc 33 24324 DNA Artificial Synthetic 243 ggtaaaggtt ggcaaaaaga taac 24 244 27 DNAArtificial Synthetic 244 gcgccgaggt cttggggtgg ttacaag 27 245 37 DNAArtificial Synthetic 245 tctcgtctcg gttttccgag actgagacct cggcgcg 37 24629 DNA Artificial Synthetic 246 cacttgcttc aggaccatat ttctctctc 29 24733 DNA Artificial Synthetic 247 cgcgccgagg acaccttttt tagggtgctt tgt 33248 22 DNA Artificial Synthetic 248 aaaatcgatg gtaaaggttg gc 22 249 22DNA Artificial Synthetic 249 agttctgcag taccggattt gc 22 250 20 DNAArtificial Synthetic 250 tcgctactag ttgcttagtg 20 251 20 DNA ArtificialSynthetic 251 gtaaacataa gcaactttag 20 252 41 DNA Artificial Synthetic252 cttgtaacca ccccaagatt atctttttgc caacctttac c 41 253 51 DNAArtificial Synthetic 253 acaaagcacc ctaaaaaagg tgtagagaga aatatggtcctgaagcaagt g 51

1. A method for detecting a target sequence, comprising: a) providing:i) a sample containing synthetic DNA amplified from genomic DNA, saidgenomic DNA suspected of containing said target sequence; ii)oligonucleotides capable of forming an invasive cleavage structure withsaid synthetic DNA when said genomic DNA contains said target sequence;and iii) an agent for detecting the presence of an invasive cleavagestructure; and b) exposing said sample to said oligonucleotides and saidagent.
 2. The method of claim 1, wherein said agent comprises a cleavageagent.
 3. The method of claim 1, wherein said genomic DNA is nickeddouble stranded genomic DNA, and wherein said synthetic DNA is amplifiedby extension of 3′ ends in said nicked double-stranded genomic DNAsuspected of containing said target sequence.
 4. The method of claim 1,wherein said synthetic DNA amplified from genomic DNA is amplified byextension of multiple primers on said genomic DNA suspected ofcontaining said target sequence.
 5. The method of claim 2, wherein saidexposing said sample to said oligonucleotides and said agent comprisesexposing said sample to said oligonucleotides and said agent underconditions wherein an invasive cleavage structure is formed between saidsynthetic DNA and said oligonucleotides if said target sequence ispresent in said genomic DNA, wherein said invasive cleavage structure iscleaved by said cleavage agent.
 6. The method of claim 5, furthercomprising the step of c) detecting said cleavage of said invasivecleavage structure.
 7. The method of claim 1, wherein said synthetic DNAcomprises a first region and a second region, said second regiondownstream of and contiguous to said first region, and wherein saidoligonucleotides comprise first and second oligonucleotides, saidwherein at least a portion of said first oligonucleotide is completelycomplementary to said first portion of said synthetic DNA and whereinsaid second oligonucleotide comprises a 3′ portion and a 5′ portion,wherein said 5′ portion is completely complementary to said secondportion of said synthetic DNA.
 8. The method of claim 7, wherein said 3′portion of said second oligonucleotide comprises a 3′ terminalnucleotide not complementary to said target nucleic acid.
 9. The methodof claim 7, wherein said 3′ portion of said second oligonucleotideconsists of a single nucleotide not complementary to said target nucleicacid.
 10. The method of claim 4, wherein said primers are randomprimers.
 11. The method of claim 4, wherein said primers are exonucleaseresistant.
 12. The method of claim 6, wherein said detecting thecleavage of said invasive cleavage structure comprises detection offluorescence.
 13. The method of claim 6, wherein said detecting thecleavage of said invasive cleavage structure comprises detection ofmass.
 14. The method of claim 6, wherein said detecting the cleavage ofsaid invasive cleavage structure comprises detection of fluorescenceenergy transfer.
 15. The method of claim 6, wherein said detecting thecleavage of said invasive cleavage structure comprises detectionselected from the group consisting of detection of radioactivity,luminescence, phosphorescence, fluorescence polarization, and charge.16. The method of claim 1, wherein said synthetic DNA is attached to asolid support.
 17. The method of claim 1 wherein said genomic DNAsuspected of containing said target sequence is attached to a solidsupport.
 18. The method of claim 7, wherein said first oligonucleotideis attached to a solid support.
 19. The method of claim 7, wherein saidsecond oligonucleotide is attached to a solid support.
 20. The method ofclaim 2, wherein said cleavage agent comprises a structure-specificnuclease.
 21. The method of claim 20, wherein said structure-specificnuclease comprises a thermostable structure-specific nuclease.
 22. Themethod of claim 20, wherein said structure specific nuclease comprises a5′ nuclease.
 23. The method of claim 22, wherein said 5′-nucleasecomprises a thermostable 5′-nuclease.
 24. The method of claim 22,wherein a portion of the amino acid sequence of said nuclease ishomologous to a portion of the amino acid sequence of a thermostable DNApolymerase derived from a thermophilic organism.
 25. The method of claim24, wherein said thermophilic organism is selected from the groupconsisting of Thermus aquaticus, Thermus flavus, and Thermusthermophilus.
 26. The method of claim 22, wherein said 5′-nucleasecomprises a FEN nuclease.
 27. The method of claim 23, wherein saidthermostable 5′ nuclease comprises a thermostable FEN nuclease.
 28. Themethod of claim 27, wherein said wherein a portion of the amino acidsequence of said nuclease is homologous to a portion of the amino acidsequence of a thermostable FEN nuclease derived from a thermophilicorganism.
 29. The method of claim 28, wherein said thermophilic organismis from an Archaebacterial species.
 30. The method of claim 29, whereinsaid Archaebacterial species is selected from the group consisting ofMethanococcus jannaschii, Pyrococcus furiosus, Pyrococcus woesei,Methanobacterium thermoautotrophicum, and Archaeoglobus fulgidus.
 31. Anucleic acid treatment kit comprising oligonucleotides capable offorming an invasive cleavage structure with synthetic DNA amplified fromgenomic DNA suspected of containing a target sequence when said genomicDNA contains a target sequence.
 32. The kit of claim 31, wherein saidsynthetic DNA amplified from genomic DNA comprising a target sequencecomprises a first region and a second region, said second regiondownstream of and contiguous to said first region, the kit comprising:a) a first oligonucleotide, wherein at least a portion of said firstoligonucleotide is completely complementary to said first portion ofsaid first target nucleic acid; and b) a second oligonucleotidecomprising a 3′ portion and a 5′ portion, wherein said 5′ portion iscompletely complementary to said second portion of said target nucleicacid.
 33. The kit of claim 32, further comprising a cleavage agent. 34.The kit of claim 32, wherein said kit further comprises a solid support.35. The kit of claim 34, wherein said first oligonucleotide is attachedto said solid support.
 36. The kit of claim 34, wherein said secondoligonucleotide is attached to said solid support.
 37. The kit of claim33, wherein said cleavage agent comprises a structure-specific nuclease.38. The kit of claim 37, wherein said structure-specific nucleasecomprises a thermostable structure-specific nuclease.
 39. The kit ofclaim 33, wherein said cleavage agent comprises a 5′ nuclease.
 40. Thekit of claim 39, wherein said 5′ nuclease comprises a thermostable 5′nuclease.
 41. The kit of claim 40, wherein a portion of the amino acidsequence of said nuclease is homologous to a portion of the amino acidsequence of a thermostable DNA polymerase derived from a thermophilicorganism.
 42. The kit of claim 41, wherein said thermophilic organism isselected from the group consisting of Thermus aquaticus, Thermus flavus,and Thermus thermophilus.
 43. The kit of claim 37, wherein saidstructure-specific nuclease comprises a FEN-1 endonuclease.
 44. The kitof claim 38, wherein said thermostable structure-specific nucleasecomprises a thermostable FEN nuclease.
 45. The kit of claim 44, whereinsaid wherein a portion of the amino acid sequence of said nuclease ishomologous to a portion of the amino acid sequence of a thermostable FENnuclease derived from a thermophilic organism.
 46. The kit of claim 45,wherein said thermophilic organism is from an Archaebacterial species.47. The kit of claim 46, wherein said Archaebacterial species isselected from the group consisting of Methanococcus jannaschii,Pyrococcus furiosus, Pyrococcus woesei, Methanobacteriumthermoautotrophicum, and Archaeoglobus fulgidus.
 48. The kit of claim31, further comprising a buffer solution.
 49. The kit of claim 48,wherein said buffer solution comprises a source of divalent cations. 50.The kit of claim 49, wherein said divalent cation is selected from thegroup consisting of Mn²⁺ and Mg²⁺ ions.
 51. The kit of claim 31, furthercomprising said target nucleic acid.
 52. The kit of claim 31, furthercomprising amplification primers.
 53. A kit for detecting the presenceof a synthetic DNA comprising a FEN-1 endonuclease and amplificationprimers.